Compositions and methods for treating diseases

ABSTRACT

The present invention provides compositions and methods of use pertaining to rAAV-mediated delivery of therapeutically effective molecules for treatment of diseases such as Pompe disease. These compositions in combination with various routes and methods of administration result in targeted expression of therapeutic molecules in specific organs, tissues and cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 13/527,350, filed Jun. 19, 2012; which is acontinuation-in-part of U.S. application Ser. No. 12/305,869, filed Apr.12, 2010; which is a §371 national phase application of InternationalApplication No. PCT/US2008/054911, filed Feb. 25, 2008, which claims thepriority benefit of U.S. Provisional Application No. 60/891,369, filedFeb. 23, 2007, all of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with U.S. government support under Grant Nos.HL59412 and NIH 5F32HL095282-03 awarded by the Heart, Lung and BloodInstitute of the National Institutes of Health. The U.S. government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the fields of molecular biology, genetherapy, and medicine. In one embodiment, the invention provides a genetherapy-based treatment for neuromuscular and lysosomal storagediseases.

BACKGROUND OF THE INVENTION

Pompe disease is both a lysosomal and glycogen storage disorderresulting from acid α-glucosidase (GAA) deficiency. GAA is normallyactive in the lysosome where it degrades excess glycogen by cleaving theα-1,4 and α-1,6 glycosidic bonds. Without adequate GAA activity, massiveamounts of glycogen accumulate in all cells. Despite systemicaccumulation of lysosomal glycogen in Pompe disease, skeletal andcardiac muscle dysfunction have been traditionally viewed as theprinciple basis for muscle weakness in this disorder.

SUMMARY OF THE INVENTION

The present invention provides rAAV vectors for delivery of therapeuticgenes into various tissues, organs, and cells including skeletal muscle,the heart, and the CNS, for restoration of neuromuscular junctionintegrity, and/or for treatment of neuromuscular diseases and glycogenstorage diseases. Advantageously, the rAAV vectors of the presentinvention provide long-term, sustained expression of the therapeuticgene of interest in a subject.

In certain embodiments, the present invention provides treatment forneuromuscular diseases including, but not limited to, Pompe disease,amyotrophic lateral sclerosis, spinal muscular atrophy, multiplesclerosis, glycogen storage disease type 1a, limb Girdle musculardystrophy, Barth syndrome, and myasthenia gravis.

In one embodiment, compositions and methods for treating lysosomalstorage diseases (e.g., glycogen storage diseases such as Pompe) aredescribed herein.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

In one embodiment, a method as described herein includes administeringto a mammalian subject having an acid alpha-glucosidase deficiency acomposition including at least one rAAV virion including apolynucleotide encoding acid alpha-glucosidase, the polynucleotideinterposed between a first AAV inverted terminal repeat and second AAVinverted terminal repeat, wherein administration of the compositionresults in increased motoneuron function in the mammalian subject. Themammalian subject can have Pompe disease. The composition can beadministered intravenously intrathecally, or intramuscularly. In oneembodiment, the at least one rAAV virion can include serotype 1, 8, 9,and/or rh10 capsid proteins. The composition can be administered to thediaphragm of the mammalian subject and travel to at least one motoneuronby retrograde transport or can be administered directly to the centralnervous system.

Another method as described herein includes administering to a mammaliansubject having Pompe disease a composition including at least one viralvector encoding acid alpha-glucosidase, wherein administration of thecomposition results in increased motoneuron function in the mammaliansubject and treats Pompe disease. The at least one viral vector can be arAAV vector.

The composition can be administered by intrathecal, intravenous,intramuscular, or parenteral route. In one embodiment, the rAAV vectorcan be within a rAAV virion including serotype 1, 8, 9, and/or rh10capsid proteins.

The composition can be administered to the diaphragm of the mammaliansubject and travel to at least one motoneuron by retrograde transport,or can be administered to the central nervous system.

Although compositions and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable compositions and methods are described below. Allpublications, patent applications, and patents mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Theparticular embodiments discussed below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1A, FIG. 1B and FIG. 1C are scans of photographs of stained hearttissues and FIG. 1D is a graph showing that intravenous delivery ofrAAV2/9 results in high-level transduction of the heart. One-day-oldC57BL6/129SvJ mouse neonates (n=5) were injected with 1×10¹¹ vg of rAAVpseudotypes AAV2/1, AAV2/8, and AAV2/9 carrying the CMV-lacZ constructvia the previously described temporal vein delivery route. At 4-weekspostinjection, the β-galactosidase enzyme detection assay was performedto quantify lacZ expression levels. FIG. 1A, FIG. 1B and FIG. 1C showX-Gal-stained cryosections from hearts injected with AAV2/1 (FIG. 1A),AAV2/8 (FIG. 1B), and AAV2/9 (FIG. 1C). FIG. 1D shows β-Galactosidaseenzyme levels in hearts (n=5).

FIG. 2A and FIG. 2B are graphs showing expression biodistributionanalysis of β-galactosidase enzyme levels detected in various specimensfollowing delivery of the rAAV2/8-CMV-lacZ or rAAV2/9-CMV-lacZconstructs. FIG. 2A shows the biodistribution across muscle tissues incomparison to myocardium. Ht indicates heart; Di, diaphragm; Qu,quadriceps; So, soleus; ED, extensor digitorum longus; TA, tibialisanterior. FIG. 2B shows the expression biodistribution in nonskeletalmuscle. Br indicates brain; Lu, lung; Sm, small intestine; Ki, kidney;Spl, spleen.

FIG. 3A, FIG. 3B and FIG. 3C are graphs showing a time-course assayfollowing rAAV2/9-mediated delivery of CMV-lacZ. FIG. 3A: followingdelivery of transgene using rAAV2/9, expression levels plateaued inskeletal muscle at 4-weeks post-administration and continued to increasein heart for the 8-week duration of the experiment. FIG. 3B shows vectorgenomes per cell also continued to increase in cardiac tissue but notskeletal muscle for the duration of the experiment. FIG. 3C shows RNAtranscripts also increased in cardiac tissue for the duration of theexperiment (n=4 per time point).

FIG. 4A is a graph showing β-galactosidase expression level analysis ofheart and skeletal muscle (4-weeks' post-administration) from mice thatwere injected with 1×10¹¹ vg of rAAV2/9-CMV-lacZ as 1-day old neonates.FIG. 4B is a graph showing β-galactosidase expression level analysis ofheart and skeletal muscle (4 weeks' post-administration) from mice thatwere injected with 1×10¹¹ vg of rAAV2/9-CMV-lacZ via the jugular vein at3 months of age (n=3).

FIG. 5A is a graph showing GAA activity in tissue specimens from thehearts of rhesus macaques intravenously injected at birth with eitherrAAV2/1-CMV-hGaa or rAAV2/9-CMV-hGaa. Y-axis shows total GAA activityminus background activity from noninjected controls per vector genomedelivered. FIG. 5B is a graph demonstrating the vector genomebiodistribution profile between heart and skeletal muscle tissue fromrhesus macaque intravenously injected at birth with rAAV2/9-CMV-hGaa.All data are at 6 months' post-vector administration.

FIG. 6A, FIG. 6B and FIG. 6C are graphs showing the results of minuteventilation (mL/min) at baseline and during 10 minutes of hypercapnia in6 month (FIG. 6A), 12 month (FIG. 6B) and >21 month (FIG. 6C) controland GAA^(−/−) mice. MEAN±SEM; *=GAA^(−/−) different from control, †=maledifferent from female.

FIG. 7A is a graph showing the results from minute ventilation atbaseline and the mean response to hypercapnia in control, GAA^(−/−) andmuscle specific GAA mice. Muscle specific GAA mice are maintained on theGAA^(−/−) background, but express GAA only in skeletal muscle. FIG. 7Bis a graph showing the results of diaphragmatic contractile function forcontrol, GAA^(−/−) and muscle specific GAA mouse diaphragm at 12 monthsof age.

FIG. 8 is a graph showing mean inspiratory flow provides an estimate ofthe neural drive to breathe. Baseline mean inspiratory flow in 6 month,12 month and >21 month control and GAA^(−/−) mice. MEAN±SEM; *=differentfrom control; no age or gender differences.

FIG. 9A is a graph and FIG. 9B is a histostain showing the results ofglycogen quantification for spinal cord segments C₃-C₅ (FIG. 9A) in6-month, 12-month and >21-month old control and GAA^(−/−) mice. Thephrenic motor pool lies within cervical spinal segments C₃-C₅.Histological glycogen detection (FIG. 9B) with the Periodic Acid Schiffstain. Phrenic motoneurons (arrows) were identified by the retrogradeneuronal tracer Fluoro-Gold® applied to the diaphragm.

FIG. 10A is a graph showing the results of a 30-sec peak amplitude ofthe moving time average for control and GAA^(−/−) mice. P_(a)CO₂ valuesare similar. FIG. 10B is a neurogram showing results from a raw phrenicneurogram (top panel) and moving time average (bottom panel) for amechanically ventilated control and GAA^(−/−) mouse with similarP_(a)CO₂ values. Scale, amplifier gain, filter settings, and recordingconfigurations were identical in the two preparations.

FIG. 11 is a graph showing that intravenous injection of rAAV2/1 leadsto clearance of glycogen in affected diaphragm tissue. One yearpost-injection, diaphragm tissue from Gaa^(−/−) mice administeredrAAV2/1-CMV-GAA intravenously and untreated age-matched controlGaa^(−/−) mice was fixed and stained with periodic acid-Schiff (PAS) bystandard methods (Richard Allen, Kalamazoo, Mich.). Photographs weretaken using a Zeiss light microscope, Olympus camera, and MagnaFire®digital recording system. Magnification ×400.

FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D are a series of graphs showingthat systemic delivery of rAAV2/1-CMV-hGAA confers improved ventilationin response to hypercapnia six months post-treatment. Ventilation oftreated Gaa^(−/−) (n=6) and age-matched untreated Gaa^(−/−) andC57BL6/129SvJ (n=10) was assessed using barometric whole-bodyplethysmography. *=p≦0.05

FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D are a series of graphs showingthat systemic delivery of rAAV2/1-CMV-hGAA confers improved ventilationin response to hypercapnia twelve months' post-treatment. Ventilation oftreated Gaa^(−/−) (n=12) and age-matched untreated Gaa^(−/−) andC57BL6/129SvJ (n=10) was assessed using barometric whole-bodyplethysmography. *=p≦0.05

FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D are a series of graphs showingthat ventilatory function is significantly improved in AAV2/1-treatedmice. Ventilatory function was assayed by awake, unrestrained, wholebody barometric plethysmography. Graphs show the minute ventilationresponse to hypercapnia over the 10-minute period of time.

FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D are a series of graphs showingthat ventilatory function is significantly improved in AAV2/1-treatedmice. Graphs show the peak inspiratory flow response to hypercapnia overthe 10-minute period of time.

FIG. 16 is a schematic illustration of a Fuller Phrenic BurstAmplitude-Hybrid. The phrenic burst amplitude measured in voltsdescribes the magnitude of the phrenic nerve with each respiration. Thelower voltage in the GAA animals indicates defective phrenic motorneuron function. This figure shows restoration of phrenic outputfollowing AAV-GAA delivery to the diaphragm.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, FIG. 17F and FIG. 17Gare a graph (FIG. 17A) and a series of photographs (FIG. 17B-FIG. 17G)of phrenic motoneurons illustrating cervical spinal cord (C₁-C₅)glycogen content. Biochemical glycogen quantification (μg glycogen/mgwet weight) of the spinal cord in control and Gaa^(−/−) mice at 6, 12and >21 months of age (FIG. 17A). *=Gaa^(−/−) different from control,p<0.01, †=6 months different from >21 months, p<0.01. Multiple motorpools exhibit positive staining for glycogen in the Gaa^(−/−) mousecervical spinal cord (FIG. 17E) vs. control (FIG. 17B). Phrenicmotoneurons were labeled with Fluoro-Gold® in control (FIG. 17C) andGaa^(−/−) (FIG. 17F) mice. Gaa^(−/−) labeled phrenic motoneuron exhibitsa more intense stain for glycogen (FIG. 17G) vs. a control phrenicmotoneuron (FIG. 17D).

FIG. 18A and FIG. 18B are a pair of graphs showing age-dependent declinein minute ventilation. Control and GAA deficient mice were evaluated forVe/VCO₂ (A) and minute ventilation (B) at 6, 12, >21 months. GAA KO micehave ½ normal Ve/VCO₂ and minute ventilation compared to controls.

FIG. 19A, FIG. 19B and FIG. 19C illustrate Minute Ventilation Responseto Hypercapnia. Minute ventilation of the 60 minute baseline (21% O₂,balanced N₂) and 10 minute response to hypercapnia (7% CO₂, balanced O₂)for 6 (FIG. 19A), 12 (FIG. 19B) and >21 (FIG. 19C) month old control andGaa^(−/−) mice. *=control different from Gaa^(−/−), p<0.01.

FIG. 20A and FIG. 20B are a pair of graphs, and FIG. 20C is a series oftracings showing Muscle-Specific hGaa Mice. Force frequency measurementsfor B6/129 (n=3), Gaa^(−/−) (n=3) and muscle specific hGaa mice (n=6)(FIG. 20A). †=Gaa^(−/−) different from control and muscle specific hGaamice. Minute ventilation at baseline and the mean response tohypercapnia for B6/129, Gaa^(−/−) and muscle specific hGaa mice(n=8/group) (FIG. 20B). *=different from control, ¥=all groups differentfrom each other. All values considered significant at p<0.01.Representative airflow tracings from un-anesthetized mice during quietbreathing (baseline) and respiratory challenge (hypercapnia) areprovided in FIG. 20C. The scaling is identical in all panels. Theairflow calibration is in mL/sec.

FIG. 21A is a graph and FIG. 21B is a series of tracings showing PhrenicInspiratory Burst Amplitude. Thirty second mean phrenic inspiratoryburst amplitude for control, Gaa^(−/−) and muscle specific hGaa micewith similar arterial P_(a)CO₂ values (shown on graph). *=different fromcontrol, p<0.01. Raw phrenic amplitude (top traces) and rectified,integrated trace (bottom traces) from representative control, Gaa^(−/−)and MTP mice are shown (scaling is identical in each panel).

FIG. 22 is a photograph of an agarose gel showing that genomic DNAisolated from diaphragm contains control gene post-vector delivery.

FIG. 23 is a photograph of a gel showing that genomic DNA isolated fromthe phrenic nucleus.

FIG. 24 is a graph showing that ventilation is improved 4 weekspost-injection with AAV-CMV-GAA (2.52×10¹⁰ particles).

FIG. 25 shows vector genome copies in the tibialis anterior (TA) andlumbar spinal cord after direct intramuscular injection of genome copiesof AAV2/9-GAA into Gaa^(−/−) animals. Briefly, Gaa^(−/−) animalsreceived a single injection of AAV2/9-DES-GAA or AAV2/9-CMV-GAA vectorsin the tibialis anterior muscle. Vector genome copies were assessed inthe tibialis anterior and lumbar spinal cord 28 days post-injection.Both constructs demonstrate efficient transduction of skeletal muscleand retrograde transport of the therapeutic transgene.

FIG. 26 shows vector genome copy and GAA enzyme activity in Pompeanimals following a single injection of AAV2/9-hGAA in the tibialisanterior muscle. Vg copies in the tibialis anterior (A) and (B) lumbarspinal cord at one month post-injection are shown. PCR data indicatessufficient retrograde transduction of AAV vectors in the spinal cord.(C) GAA activity levels at the site of injection (TA) result in asignificant increase in enzymatic level in Gaa−/− mice.

FIG. 27 shows staining of neuromuscular junction (NMJ) following asingle injection of AAV2/9-DES-hGAA in the tibialis anterior.Immunostaining of the NMJ shows that rAAV2/9 mediated delivery of hGAAresults in the reversal of pathology of Pompe disease and restoration ofthe NMJ in treated Gaa^(−/−) animals.

FIG. 28 shows transduction of phrenic motor neuron pool after directintraspinal injection of AAV reporter constructs in mouse C3-C5 region.(A) Positive detection of AAV reporter constructs (*) at the site ofinjection in fixed, intact brain and spinal cord preparations. (B)Cross-sectional epifluorescent detection of AAV-GFP demonstratestransduction of the phrenic motor pool.

FIG. 29 shows immunohistochemistry detection of GAA protein expressionin phrenic motor neuron pool after direct intraspinal injection ofAAV-GAA in the mouse C3-C5 region. Detection of AAV-GAA in the phrenicmotorneuron pool is shown (A) at low and (B) at higher magnification.Dark brown staining is positive for GAA protein detection.

FIG. 30 shows intrathoracic injection of infrared dye in mouse. Imagingindicates positive detection of the dye across the surface of thediaphragm.

FIG. 31 shows diaphragm and phrenic motor neuron activity duringhypercapnic conditions after intrathoracic injection of AAV vectors.Untreated Gaa^(−/−) animals show a weak and blunted EMG (diaphragm) andburst amplitude (phrenic nerve) compared to AAV2/9-CMV-GAA andAAV2/9-DES-GAA treated animals.

FIG. 32 shows detection of AAV-reporter in the cervical and thoracicspinal cord. Anti-GFP immunohistochemical staining detects positivephrenic (left) and costal (right) motor neuron staining demonstratingintrathoracic administration of AAV2/9 results in retrogradetransduction.

FIG. 33 shows diaphragm EMG (A), copies of AAV genome vectors (B), andimprovement in phrenic nerve signal propagation (C) followingintrathoracic administration of rAAV2/9-CMV-GAA or rAAV2/9-DES-GAAvectors into Gaa^(−/−) animals.

FIG. 34 shows correction of pathological symptoms in Gaa^(−/−) animalsafter intravenous administration of AAV2/9-CMV-GAA or AAV2/9-DES-GAAvectors. (A) shows ejection fraction at three months post-injection.AAV-DES treated animals exhibit significant improvement in ejectionfraction. Mean and sem (n=6). (B) shows that all treatments resulted ina significant increase in body weight at one and three months postinjection. (C) shows PR intervals at one month and three months posttreatment. (D) shows improvement of cardiac function post intravenousinjection. (E) shows GAA enzymatic activity in the heart afterintravenous administration. (F) In vitro force-frequency measurements ofdiaphragm of AAV2/9-CMV and AAV2/9-DES-GAA treated animals exhibitincreased max titanic force. Mean and sem (n=6). *P value<0.05 ascompared to untreated mice. The AAV2/9-GAA treatment results in animprovement of contractile function at frequencies above 60 Hz. (G)shows GAA enzyme activity in respiratory muscle. (H) shows vector genomecopies in the cardiac, diaphragm, and spinal cord after intravenousadministration of AAV2/9-CMV and AAV2/9-DES-GAA vectors. (I) shows theGAA expression level after intravenous administration of AAV2/9-CMV andAAV2/9-DES-GAA vectors.

FIG. 35 is a schematic representation of AAV and HSV viruses. Wild-typeand recombinant genomes are shown with major genetic elements.

FIG. 36 illustrates the production of AAV by HSV coinfection methodusing adherent cells. 1 and 2) generation of rHSV stocks; 3) coinfectionwith rHSV; 4) harvest and recovery of rAAV.

FIG. 37 shows streamlined purification of rAAV2/9 by pI precipitationand column chromatography. (A) Silver stained PAGE of crude lysate (lane1), crude lysate after microfluidization (lane 2), AAV2/9-retainingsupernatant after pI precipitation (lane 3); (B) Silver stained PAGE ofpI-purified rAAV2/9 containing fractions (lanes 2 to 7) after SPSepharose IEC and concentration.

FIG. 38 is an evaluation of cross-sections of the sciatic nerve inwild-type and Gaa^(−/−) mice. Gaa^(−/−) mice display irregularmorphometry and an increase in the amount of extracellular matrix.

FIG. 39 shows longitudinal nerve section stained for H&E. Comparison ofwild-type and Gaa^(−/−) mice reveals an increase in nuclear stainingindicates dedifferentiation and proliferation of Schwann cells.

FIG. 40 is an evaluation of neuromuscular junction integrity of thesoleus in wild-type and Gaa^(−/−) mice. (A) Longitudinal sections fromsoleus muscle. Note the marked changes in Gaa^(−/−) where dissociationof the neuromuscular junction is evident. (B) Neuromuscular junction inwhole mount of mouse diaphragm. Typical intact neuromuscular junction(arrow) and atypical neuromuscular junction (*) in the Gaa^(−/−)diaphragm. Alpha-bungarotoxin (red) and NeurofilamentH (green).

FIG. 41 shows morphometric analysis of the diaphragm NMJ. Gaa^(−/−)acetylcholine receptors (AcR) are significantly larger compared to WT.Note the apparent focal and non-congruent appearance of theα-bungarotoxin labeling in the Gaa^(−/−).

FIG. 42 shows cross-sections of sciatic nerve in WT and Gaa mice.Gaa^(−/−) mice display irregular axonal morphometry and an increase inextracellular matrix.

FIG. 43 shows the loss of Synaptotagmin at the NMJ. Gaa^(−/−) animals(B) exhibit a profound loss of synaptotagmin expression when compared toWT (A). Magenta-NFH.

FIG. 44 shows an experimental design of rAAV-mediated expression of GAAin animals.

FIG. 45 shows a decrease in the size of acetylcholine receptors in theleg of the Gaa^(−/−) mouse injected with AAV2/9-DESMIN-GAA. Gaa^(−/−)animals show a significant increase in AChR size, whereas a decrease inthe size of AChR after AAV2/9-DESMIN-GAA treatment provides positivetherapeutic effects against NMJ pathology. In a 23-month-old Gaa^(−/−)mouse, two months after the injection of the AAV2/9-DESMIN-GAA vectorsdirectly into the right tibialis anterior muscle, acetylcholine receptor(AChR) size in the injected leg and contralateral leg are measured.

FIG. 46 shows that Gaa^(−/−) animals exhibit a loss of NMJ integritywhen compared to wild-type (WT) animals. Normalization of the junctionappears to occur at 1 month post AAV9-GAA administration (right panel).

FIG. 47 shows axonal labeling (Gap43) in AAV2/9-GAA treated Gaa^(−/−)animals.

FIG. 48 shows ventilation in Gaa^(−/−) mice after intraspinal deliveryof AAV-GAA. Vector treated animals (dashed line) displayed improvedventilatory parameters during baseline (left panels) and hypercapnicrespiratory challenge (right panels).

DETAILED DESCRIPTION

The present invention provides rAAV vectors for delivery of therapeuticgenes into various tissues, organs, and cells including skeletal muscle,the heart, and the CNS, for restoration of neuromuscular junctionintegrity, and/or for treatment of neuromuscular diseases.Advantageously, the rAAV vectors of the present invention providelong-term, sustained expression of the therapeutic gene of interest in asubject.

In one embodiment, the present invention provides a recombinantadeno-associated virus (AAV) virion, comprising a rAAV vector thatcomprises a heterologous nucleic acid molecule (also referred to as atransgene or a therapeutic gene), and the AAV vector is encapsidated bya viral capsid.

In one specific embodiment, the rAAV virion comprises a rAAV vector thatcomprises a heterologous nucleic acid molecule (also referred to as atransgene or a therapeutic gene) encoding a protein or polypeptide ofinterest,

wherein the heterologous nucleic acid molecule is operably linked tocontrol elements (e.g., promoter, enhancer) capable of directing in vivoor in vitro expression of the protein or polypeptide of interest,

wherein the heterologous nucleic acid molecule is flanked on each endwith an AAV inverted terminal repeat, and

wherein the rAAV vector is encapsidated by a viral capsid.

In a preferred embodiment, the present invention pertains to pseudotypedrAAV vectors. In certain embodiments, the present invention pertains torAAV2/x vectors, which comprise the vector genome of AAV2 and capsids ofAAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10).In preferred embodiments, the present invention pertains to rAAV2/1(also referred to in the Figures as rAAV1), rAAV2/5, rAAV2/8 (alsoreferred to in the Figures as rAAV8), and rAAV2/9 (also referred to inthe Figures as rAAV9) vectors.

In one embodiment, the rAAV vector comprises a cytomegalovirus (CMV)promoter. In another embodiment, the rAAV vector comprises a desmin(DES) promoter. In one specific embodiment, the rAAV vector comprisesregulatory elements for tissue-specific expression of the transgene,such as for example, myocyte specific enhancer factor 2 (MEF2) and myoDenhancer element.

In another embodiment, the present invention provides a method fortreating a neuromuscular disease, comprising administering to a subjectin need of such treatment, an effective amount of a compositioncomprising a rAAV vector of the present invention.

In one specific embodiment, the present invention provides a method forrestoring neuromuscular junction integrity and/or for improving impairedneuromuscular junction integrity in a subject, wherein the methodcomprises administering to the subject, an effective amount of acomposition comprising a rAAV vector of the present invention. Incertain embodiments, the impaired neuromuscular integrity is caused byneuromuscular disease or injury.

In another embodiment, the present invention provides a method fortreating a disease by delivery of a therapeutic gene into cells ofinterest, wherein the method comprises introducing into a cell, aneffective amount of a composition comprising a rAAV vector of thepresent invention.

In certain embodiments, the rAAV vector of the present invention isadministered via intravenous, intramuscular, intrathoracic, intrathecal,intracisternal, or intraspinal injection. In certain embodiments, therAAV vectors are administered to the skeletal muscle, diaphragm, costal,and/or cardiac muscle cells of a subject. In certain embodiments, therAAV vectors are delivered to neuronal cells in the peripheral and/orcentral nervous system via direct or retrograde transport.

In certain embodiments, the present invention provides treatment forneuromuscular diseases including, but not limited to, Pompe disease,amyotrophic lateral sclerosis, spinal muscular atrophy, multiplesclerosis, glycogen storage disease type 1a, limb Girdle musculardystrophy, Barth syndrome, and myasthenia gravis.

In one embodiment, the rAAV vector comprises a therapeutic gene encodingGAA and the rAAV vector is delivered into the central nervous system(e.g., the spinal cord and the brain) to reduce glycogen accumulation inthe central nervous system in a subject with Pompe disease.

In another embodiment, the present invention provides treatment for aneuromuscular disease that is not Pompe disease. In another embodiment,the present invention improves impaired neuromuscular junction integrityin a subject that does not have Pompe disease. The animal model of Pompedisease is used as an example to illustrates that the rAAV vectors ofthe present invention result in effective delivery of the therapeuticgene encoding GAA into cells, tissues and organs of interest. The animalmodel of Pompe disease is also used as an example to illustrate that therAAV vectors of the present invention result in improvement ofneuromuscular junction integrity. One skilled in the art, in view ofthese examples, would readily recognize that the rAAV vectors of thepresent invention can also be used to deliver other therapeutic genes tocells, tissues and organs of interest (e.g., neuronal cells), therebyproviding treatment for neuromuscular diseases and/or improvingneuromuscular junction integrity in a subject that does not have Pompedisease.

In certain embodiments, compositions and methods including rAAV virionshaving rAAV vectors expressing GAA for treating a mammalian subjecthaving a GAA deficiency are described herein. In one embodiment,compositions comprising rAAV serotypes (e.g., serotypes 1-10, orderivatives thereof) expressing therapeutic molecules in combinationwith an intravenous route of administration results in rAAV serotypesthat are more readily able to cross the vasculature and efficientlytransduce a particular tissue type (e.g., cardiac tissue, diaphragmtissue, central nervous system tissue.

In another embodiment, the AAV are modified to include ligands which arecell and/or tissue specific so that the compositions are administeredsystemically and absorption into targets is directed and specific.

When taking into consideration those characteristics desirable in avehicle for gene delivery in a mammal, the (4.7-kb) nonpathogenicparvovirus rAAV emerged as an attractive choice, mainly because of itssmall size and proven ability to persist for long periods of time ininfected cells. rAAV is a single-stranded DNA virus that requires ahelper, such as herpes virus or adenovirus, to replicate. With recentdiscoveries of additional serotypes of rAAV, it has become possible toselect those with the most advantageous tropisms to target and/or evadetissues as desired for specific applications. The most optimalgene-delivery system for any therapeutic application will combine aclinically advantageous physical delivery route with the rAAV serotypethat has the highest natural affinity for a specifically targeted tissueof interest.

In a typical embodiment, a composition includes a rAAV virion having arAAV vector encoding GAA that improves phrenic nerve function in amammalian subject having a GAA deficiency (e.g., Pompe disease). TherAAV virion can be directly transduced into the central nervous system,or can be transduced into other tissue types (e.g., diaphragm) andtransported to the central nervous system via retrograde transport. Byimproving phrenic nerve activity in a mammalian (e.g., human) subjecthaving a GAA deficiency, resulting respiratory deficits may be corrected(e.g., reduced ventilation, reduced cardiac function, etc.)

In another embodiment, administration of the rAAV is performed viaintravenous administration (e.g., systemic delivery). In a typicalembodiment, systemic delivery is used, as it impacts cardiac, muscle andrespiratory aspects of the disease.

Other examples of different therapeutic molecules for treating lysosomalstorage diseases (LSDs) include without limitation: Hurler disease:α-L-iduronidase; Hunter disease: iduronate sulfatase; Sanfilippo:heparan N-sulfatase; Morquio A: galactose-6-sulfatase; Morquio B:acid-β-galactosidase; Sly disease: β-glucoronidase: I-cell disease:N-acetylglucosamine-1-phosphotransferase; Schindler disease:α-N-acetylgalactosaminidase (α-galactosidase B); Wolman disease: acidlipase; Cholesterol ester: acid lipase; storage disease; Farber disease:lysosomal acid ceramidase; Niemann-Pick disease: acid sphingomyelinase;Gaucher's disease: β-glucosidase (glucocerebrosidase); Krabbe disease:galactosylceramidase; Fabry disease: α-galactosidase A; GM1gangliosidosis: acid β-galactosidase; Galactosialidosis: β-galactosidaseand neuraminidase; Tay-Sach's disease: hexosaminidase A; Sandhoffdisease: hexosaminidase A and B; Neuronal Ceroid: Palmitoyl ProteinThioesterase (PPT); Neuronal Ceroid: Tripeptidyl Aminopeptidase II(TPP-I). Heterologous nucleic acid molecules or transgenes encoding thetherapeutic molecules of interest can be inserted into to the rAAVvectors of the invention for treatment of lysosomal storage diseases.

Glycogen storage disease type II (GSD II; Pompe disease; acid maltasedeficiency) is caused by deficiency of the lysosomal enzyme acidα-glucosidase (acid maltase). Three clinical forms are distinguished:infantile, juvenile and adult. Infantile GSD II has its onset shortlyafter birth and presents with progressive muscular weakness and cardiacfailure. This clinical variant is fatal within the first two years oflife. Symptoms in adult and juvenile patients occur later in life, andskeletal muscles and neurons are primarily involved. The patientseventually die due to respiratory insufficiency. Patients mayexceptionally survive for more than six decades. There is a goodcorrelation between the severity of the disease and the residual acidα-glucosidase activity, the activity being 10-20% of normal in lateonset and less than 2% in early onset forms of the disease.

Pompe disease is an inborn error of metabolism with deficiency of thelysosomal glycogen degrading enzyme acid α-glucosidase (GAA), whichultimately results in glycogen accumulation in all tissues, especiallystriated muscle. Historically, muscle weakness has been viewed as themajor contributor to respiratory deficiency in the patient population,yet other mechanisms have not been investigated. To further evaluatecontributing mechanisms of respiratory insufficiency, an animal model ofPompe disease, the Gaa^(−/−) mouse model, was used. Ventilation wasquantified in Gaa^(−/−) and control mice during quiet breathing andhypercapnia. All ventilation variables were attenuated in Gaa^(−/−) miceat 6, 12 and >21 months of age and were accompanied by elevated glycogencontent of the cervical spinal cord (C₃-C₅). Transgenic mice that onlyexpress Gaa in skeletal muscle had minute ventilation similar toGaa^(−/−) although diaphragmatic muscle function was normal,demonstrating that a mechanism other than muscle dysfunction wascontributing to ventilation impairments. Efferent phrenic nerveinspiratory burst amplitude (mV) was lower in Gaa^(−/−) mice (5.2±1.2mV) compared to controls (49.7±13.9 mV) with similar P_(a)CO₂ levels(53.1±1.2 vs. 52.2±1.4 mmHg). The data indicate that neural control ofventilation is deficient in Pompe disease and support the followingconclusions: 1) Gaa^(−/−) mice recapitulate clinical GSDII respiratorydeficits, 2) spinal glycogen accumulation may impair motor output, and3) respiratory neural control may be impaired in GSDII.

Infantile forms of Pompe have a rapid development of cardiomyopathy anddisplay myopathy and neuorpathy leading to death typically in the firstyear of life. Using periodic acid-Schiff's reagent (PAS) staining toassess diaphragm sections in mice, the progression of glycogendeposition in Gaa^(−/−) animals is examined. At 12 months of agewidespread and diffuse glycogen deposits are apparent in diaphragmsections of Gaa^(−/−) animals. Not only is there a decline indiaphragmatic contractile properties in Gaa^(−/−) animals, there is alsoa progressive worsening of contractile function with age. In vitro forcefrequency measurements illustrate a progressive loss of contractilefunction in Gaa^(−/−) mice at 3, 6, and 12 months of age. The deficiencyin GAA leads to inefficient glycogen clearance, leading to a disruptionof cellular morphology and function. Biopsies of Pompe patients alsoshow vacuolization, lysosomal glycogen accumulation and consequentrespiratory muscle weakness.

In addition, the effect of glycogen accumulation within the centralnervous system and its effect on skeletal muscle function have beenrecently discovered. The present inventors have detected substantialglycogen deposition within the Gaa^(−/−) mouse spinal cord.Specifically, PAS staining shows the degree and localization of glycogenin the motor neuron in cross-sections of spinal cord. The motor neuronappears to be swollen in some cases and varies in the degree across thesection. Phrenic nerve recordings in wild-type and Gaa^(−/−) animalsdisplay obvious discrepancies in the level of burst amplitude uponhypercapnic challenge. The inventors also discovered that amuscle-specific GAA mouse (expressed GAA in skeletal muscle but not inthe CNS) still showed functional respiratory deficits duringplethysmography measurements. The results indicate CNS-mediatedrespiratory dysfunction in Gaa^(−/−) animals and in Pompe patients.

A remarkable characteristic of Pompe disease occurring in the animalmodel is the severe kyphosis as a result of the drastic skeletalmyopathy. Gaa^(−/−) animals develop severe kyphosis at about 9-12 monthsof age. The development of neutropenia in Pompe affected individuals hasbeen described and this is apparent in the animal model where muscleweakness ultimately affects nutrient intake. Gaa^(−/−) mice treated withAAV-CMV-GAA do not develop kyphosis. With the preservation of skeletalmuscle mass and strength, treated animals are able to maintain adequatenutritional intake and therefore function similar to wild-type mice.

The Gaa^(−/−) mouse model shows the progressive pathology associatedwith glycogen storage in the CNS and skeletal muscle. The presentinventors have reported the specific role of the CNS contribution torespiratory dysfunction in the murine model of Pompe disease. In otherneuromuscular disorders, adaptations in the NMJ and motor neuron occuras a result of CNS or skeletal muscle abnormalities. It is reported thatfatigue is evident in late-onset Pompe patients and may be caused bydeficiencies that are neural-based but for the majority remain centeredaround skeletal muscle defects.

In accordance with the present invention, sciatic nerves were harvestedfrom 18 month old wild-type or Gaa^(−/−) animals. As shown in FIG. 38,nerve cross-sections from Gaa^(−/−) animals appear to vary in the amountand thickness of myelin, the diameter or axons, and show an increase inthe amount of extracellular matrix. In addition, longitudinal sectionsof sciatic nerve from Gaa^(−/−) animals display an increase in nuclearstaining, indicting dedifferentiation and proliferation of Schwann cells(FIG. 39). The staining patterns of the sciatic nerve samples in Pompedisease are also observed in traditional neuromuscular diseases such asamyotrophic lateral sclerosis, spinal muscular atrophy, multiplesclerosis, glycogen storage disease type 1a, limb Girdle musculardystrophy, Barth syndrome, and myasthenia gravis.

The fatigue present in patients with neuromuscular diseases could be dueto impairments in E-C coupling and other mechanisms. Upon comparison ofthe neuromuscular junction in longitudinal sections from the soleus andwhole mount diaphragm preparations (FIG. 40), the present inventorsnoted abrupt changes in association of the neuromuscular junction. Theincorporation of whole-mount diaphragm preparations provides an in-depthpicture of the overall status of neuromuscular junctions. Diaphragmpreparations from Gaa^(−/−) animals display an advanced and cleardistortion of junctions compared to wild-type diaphragm.

Gaucher's disease is an autosomal recessive lysosomal storage disordercharacterized by a deficiency in a lysosomal enzyme, glucocerebrosidase(“GCR”), which hydrolyzes the glycolipid glucocerebroside. In Gaucher'sdisease, deficiency in the degradative enzyme causes the glycolipidglucocerebroside, which arises primarily from degradation ofglucosphingolipids from membranes of white blood cells and senescent redblood cells, to accumulate in large quantities in the lysosome ofphagocytic cells, mainly in the liver, spleen and bone marrow. Clinicalmanifestations of the disease include splenomegaly, hepatomegaly,skeletal disorders, thrombocytopenia and anemia. For example, see U.S.Pat. No. 6,451,600.

Tay-Sachs disease is a fatal hereditary disorder of lipid metabolismcharacterized especially in CNS tissue due to deficiency of the A(acidic) isozyme of β-hexosaminidase. Mutations in the HEXA gene, whichencodes the a subunit of β-hexosaminidase, cause the A isozymedeficiency. Tay-Sachs disease is a prototype of a group of disorders,the GM2 gangliosidosis, characterized by defective GM2 gangliosidedegradation. The GM2 ganglioside (monosialylated ganglioside 2)accumulates in the neurons beginning in the fetus. GM1 gangliosidosis iscaused by a deficiency of β-galactosidase, which results in lysosomalstorage of GM1 ganglioside (monosialylated ganglioside 1). Sandhoffdisease results from a deficiency of both the A and B (basic) isozymesof β-hexosaminidase. Mutations in the HEXB gene, which encodes the βsubunit of β-hexosaminidase, cause the B isozyme deficiency.

Another LSD results from a genetic deficiency of thecarbohydrate-cleaving, lysosomal enzyme α-L-iduronidase, which causesmucopolysaccharidosis I (MPS I) (E. F. Neufeld and J. Muenzer, 1989;U.S. Pat. No. 6,426,208). See also “The mucopolysaccharidoses” in TheMetabolic Basis of Inherited Disease (C. R. Scriver, A. L. Beaudet, W.S. Sly and D. Valle, Eds.), pp. 1565-1587, McGraw-Hill, New York. In asevere form, MPS I is commonly known as Hurler syndrome and isassociated with multiple problems such as mental retardation, cloudingof the cornea, coarsened facial features, cardiac disease, respiratorydisease, liver and spleen enlargement, hernias, and joint stiffness.Patients suffering from Hurler syndrome usually die before age 10. In anintermediate form known as Hurler-Scheie syndrome, mental function isgenerally not severely affected, but physical problems may lead to deathby the teens or twenties. Scheie syndrome is the mildest form of MPS Iand is generally compatible with a normal life span, but jointstiffness, corneal clouding and heart valve disease cause significantproblems.

Fabry disease is an X-linked inherited lysosomal storage diseasecharacterized by symptoms such as severe renal impairment,angiokeratomas, and cardiovascular abnormalities, including ventricularenlargement and mitral valve insufficiency (U.S. Pat. No. 6,395,884).The disease also affects the peripheral nervous system, causing episodesof agonizing, burning pain in the extremities. Fabry disease is causedby a deficiency in the enzyme α-galactosidase A (α-gal A), which resultsin a blockage of the catabolism of neutral glycosphingolipids, andaccumulation of the enzyme's substrate, ceramide trihexoside, withincells and in the bloodstream. Due to the X-linked inheritance pattern ofthe disease, essentially all Fabry disease patients are male. Although afew severely affected female heterozygotes have been observed, femaleheterozygotes are generally either asymptomatic or have relatively mildsymptoms largely limited to a characteristic opacity of the cornea. Anatypical variant of Fabry disease, exhibiting low residual α-gal Aactivity and either very mild symptoms or apparently no other symptomscharacteristic of Fabry disease, correlates with left ventricularhypertrophy and cardiac disease. It has been speculated that reductionin α-gal A may be the cause of such cardiac abnormalities.

I-cell disease is a fatal lysosomal storage disease caused by theabsence of mannose-6-phosphate residues in lysosomal enzymes.N-acetylglucosamine-1-phospho-transferase is necessary for generation ofthe M6P signal on lysosomal proenzymes.

LSDs which affect the central nervous system require that thereplacement enzyme cross the BBB. To accomplish this, the source of thereplacement enzyme may be placed within the brain of the subject,thereby bypassing the BBB. Thus, glial progenitor cells are idealtherapeutic delivery vehicles because of their exceptional capacity tomultiply, migrate and differentiate into oligodendrocyte and astrocytesubtypes. Thus, LSDs that affect the central nervous system may betreated in a variety of manners, including genetically encoding glialprogenitor cells to secrete lysosomal proenzymes, for example, lysosomalproenzymes, and delivering the cells to damaged tissues and/or replacingthe defective cells.

In another embodiment, the compositions of the instant invention areused to treat neurological disorders. A “neurological disorder” refersto any central nervous system (CNS) or peripheral nervous system (PNS)disease that is associated with neuronal or glial cell defects includingbut not limited to neuronal loss, neuronal degeneration, neuronaldemyelination, gliosis (i.e., astrogliosis), or neuronal orextraneuronal accumulation of aberrant proteins or toxins (e.g.,β-amyloid, or α-synuclein). The neurological disorder can be chronic oracute.

Exemplary neurological disorders include but are not limited toGaucher's disease and other LSDs including Fabry disease, Tay-Sachsdisease, Pompe disease, and the mucopolysaccharidoses; Parkinson'sdisease; Alzheimer's disease; Amyotrophic Lateral Sclerosis (ALS);Multiple Sclerosis (MS); Huntington's disease; Fredrich's ataxia; MildCognitive Impairment; and movement disorders (including ataxia, cerebralpalsy, choreoathetosis, dystonia, Tourette's syndrome, kernicterus);tremor disorders, leukodystrophies (including adrenoleukodystrophy,metachromatic leukodystrophy, Canavan disease, Alexander disease,Pelizaeus-Merzbacher disease); neuronal ceroid lipofucsinoses; ataxiatelangectasia; and Rett syndrome. This term also includescerebrovascular events such as stroke and ischemic attacks.

The term “subject,” as used herein, describes an organism, includingmammals such as primates. Mammalian species that can benefit from thesubject methods include, but are not limited to, apes, chimpanzees,orangutans, humans, monkeys; and other animals such as dogs, cats,horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs,and hamsters. Typically, the subject is a human.

In some embodiments, subjects with a “neurological disorder” includesubjects at risk of developing a neurological disorder, disease orcondition and/or subjects already diagnosed with a neurologicaldisorder, disease or condition.

The injection of an rAAV vector (such as, rAAV2/9) comprising a nucleicacid molecule encoding GAA can result in a decrease in the size of AChRsize in Gaa^(−/−) animals; therefore, the rAAV-mediated delivery of GAAcan be used to restore or enhance neuromuscular transmission between thephrenic nerve and diaphragm. In one embodiment, in addition to theadministration of an AAV vector (such as, AAV2/9) comprising a nucleicacid molecule encoding GAA, one or more acetylcholinesterase inhibitors(ACI) can also be co-administered separately or simultaneously toimprove neurotransmitter release in subjects with Pompe disease.

Various acetylcholinesterase inhibitors (ACI) are known in the art,including, but not limited to, tetrahydroaminoacridine, donepezil,galantamine, rivastigmine tacrine, metrifonate, and huperzine-A (seeU.S. Patent Application Publication No. 2013/0131110).

Therapeutic Molecules Nucleic Acids for Modulating GAA Expression:

As an example, GAA is used to illustrate the invention. However,depending on the diseases, the therapeutic molecule can be substituted(infra). Transfer of a functional GAA protein into a cell or animal isaccomplished using a nucleic acid that includes a polynucleotideencoding the functional GAA protein interposed between two AAV ITRs. TheGAA-encoding polynucleotide sequence can take many different forms. Forexample, the sequence may be a native mammalian GAA nucleotide sequencesuch as one of the mouse or human GAA-encoding sequences deposited withGenbank as accession numbers NM_(—)008064, NM_(—)000152, X55080, X55079,M34425, and M34424. The GAA-encoding nucleotide sequence may also be anon-native coding sequence which, as a result of the redundancy ordegeneracy of the genetic code, encodes the same polypeptide as does anative mammalian GAA nucleotide sequence. Other GAA-encoding nucleotidesequences within the invention are those that encode fragments, analogs,and derivatives of a native GAA protein. Such variants may be, e.g., anaturally occurring allelic variant of a native GAA-encoding nucleicacid, a homolog of a native GAA-encoding nucleic acid, or anon-naturally occurring variant of native GAA-encoding nucleic acid.These variants have a nucleotide sequence that differs from nativeGAA-encoding nucleic acid in one or more bases. For example, thenucleotide sequence of such variants can feature a deletion, addition,or substitution of one or more nucleotides of a native GAA-encodingnucleic acid. Nucleic acid insertions are generally of about 1 to 10contiguous nucleotides, and deletions are generally of about 1 to 30contiguous nucleotides. In most applications of the invention, thepolynucleotide encoding a GAA substantially maintains the ability toconvert phenylalanine to tyrosine.

The GAA-encoding nucleotide sequence can also be one that encodes a GAAfusion protein. Such a sequence can be made by ligating a firstpolynucleotide encoding a GAA protein fused in frame with a secondpolynucleotide encoding another protein (e.g., one that encodes adetectable label). Polynucleotides that encode such fusion proteins areuseful for visualizing expression of the polynucleotide in a cell.

In order to facilitate long term expression, the polynucleotide encodingGAA is interposed between AAV inverted terminal repeats (ITRs) (e.g.,the first and second AAV ITRs). AAV ITRs are found at both ends of a WTAAV genome, and serve as the origin and primer of DNA replication. ITRsare required in cis for AAV DNA replication as well as for rescue, orexcision, from prokaryotic plasmids. The AAV ITR sequences that arecontained within the nucleic acid can be derived from any AAV serotype(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or can be derived from more thanone serotype. For use in a vector, the first and second ITRs shouldinclude at least the minimum portions of a WT or engineered ITR that arenecessary for packaging and replication.

In addition to the AAV ITRs and the polynucleotide encoding GAA, thenucleic acids of the invention can also include one or more expressioncontrol sequences operatively linked to the polynucleotide encoding GAA.Numerous such sequences are known. Those to be included in the nucleicacids of the invention can be selected based on their known function inother applications. Examples of expression control sequences includepromoters, insulators, silencers, response elements, introns, enhancers,initiation sites, termination signals, and pA tails.

To achieve appropriate levels of GAA, any of a number of promoterssuitable for use in the selected host cell may be employed. For example,constitutive promoters of different strengths can be used. Expressionvectors and plasmids in accordance with the present invention mayinclude one or more constitutive promoters, such as viral promoters orpromoters from mammalian genes that are generally active in promotingtranscription. Examples of constitutive viral promoters include theHerpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus(RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad EIAand cytomegalovirus (CMV) promoters. Examples of constitutive mammalianpromoters include various housekeeping gene promoters, as exemplified bythe β-actin promoter. As described in the examples below, the chickenbeta-actin (CB) promoter has proven to be a particularly usefulconstitutive promoter for expressing GAA.

Inducible promoters and/or regulatory elements may also be contemplatedfor use with the nucleic acids of the invention. Examples of suitableinducible promoters include those from genes such as cytochrome P450genes, heat shock protein genes, metallothionein genes, andhormone-inducible genes, such as the estrogen gene promoter. Anotherexample of an inducible promoter is the tetVP16 promoter that isresponsive to tetracycline.

Promoters useful according to the present invention also include, butare not limited to, neuron-specific promoters, such as synapsin I (SYN)promoter; muscle creatine kinase (MCK) promoters; cytomegalovirus (CMV)promoters; and desmin (DES) promoters. In one embodiment, theAAV-mediated expression of heterologous nucleic acids (such as humanGAA) can be achieved in neurons via a Synapsin promoter or in skeletalmuscles via a MCK promoter.

Tissue-specific promoters and/or regulatory elements are useful incertain embodiments of the invention. Examples of such promoters thatmay be used with the expression vectors of the invention include (1)creatine kinase, myogenin, alpha myosin heavy chain, human brain andnatriuretic peptide, specific for muscle cells, and (2) albumin,alpha-1-antitrypsin, hepatitis B virus core protein promoters, specificfor liver cells.

The invention also includes methods and compositions thereof which canbe used to correct or ameliorate a gene defect caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of the protein. This may be desirable when the sizeof the DNA encoding the protein subunit is large, e.g., for animmunoglobulin or the platelet-derived growth factor receptor. In orderfor the cell to produce the multi-subunit protein, a cell would beinfected with rAAV expressing each of the different subunits.

Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene would include the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribosome entry site (IRES). The use of IRES permits thecreation of multigene or polycistronic mRNAs. IRES elements are able tobypass the ribosome scanning model of 5′ methylated cap-dependenttranslation and begin translation at internal sites. For example, IRESelements from hepatitis C and members of the picornavirus family (e.g.,polio and encephalomyocarditis) have been described, as well an IRESfrom a mammalian mRNA. IRES elements can be linked to heterologous openreading frames. By virtue of the IRES element, each open reading frameis accessible to ribosomes for efficient translation. Thus, multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message. This is particularly useful when the sizeof the DNA encoding each of the subunits is sufficiently small that thetotal of the DNA encoding the subunits and the IRES is no greater thanthe maximum size of the DNA insert that the virus can encompass. Forinstance, for rAAV, the insert size can be no greater than approximately4.8 kilobases; however, for an adenovirus which lacks all of its helperfunctions, the insert size is approximately 28 kilobases.

Useful gene products include hormones and growth and differentiationfactors including, without limitation, insulin, glucagon, growth hormone(GH), parathyroid hormone (PTH), calcitonin, growth hormone releasingfactor (GRF), thyroid stimulating hormone (TSH), adrenocorticotropichormone (ACTH), prolactin, melatonin, vasopressin, β-endorphin,met-enkephalin, leu-enkephalin, prolactin-releasing factor,prolactin-inhibiting factor, corticotropin-releasing hormone,thyrotropin-releasing hormone (TRH), follicle stimulating hormone (FSH),luteinizing hormone (LH), chorionic gonadotropin (CG), vascularendothelial growth factor (VEGF), angiopoietins, angiostatin,endostatin, granulocyte colony stimulating factor (GCSF), erythropoietin(EPO), connective tissue growth factor (CTGF), basic fibroblast growthfactor (bFGF), bFGF2, acidic fibroblast growth factor (aFGF), epidermalgrowth factor (EGF), transforming growth factor a (TGFα),platelet-derived growth factor (PDGF), insulin-like growth factors I andII (IGF-I and IGF-II), any one of the transforming growth factor β(TGFβ) superfamily comprising TGFβ, activins, inhibins, or any of thebone morphogenic proteins (BMP) BMPs 1 15, any one of theheregulin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurtuin, persephin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the immunesystem including, without limitation, cytokines and lymphokines such asthrombopoietin (TPO), interleukins (IL) IL-1α, IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, and IL-17, monocyte chemoattractant protein (MCP-1), leukemiainhibitory factor (LIF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocytecolony stimulating factor (M-CSF), Fas ligand, tumor necrosis factors αand β (TNFα and TNFβ), interferons (IFN) IFN-α, IFN-β, and IFN-γ, stemcell factor, flk-2/flt3 ligand. Gene products produced by the immunesystem are also encompassed by this invention. These include, withoutlimitations, immunglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineered MHC moleculesincluding single chain MHC molecules. Useful gene products also includecomplement regulatory proteins such as membrane cofactor protein (MCP),decay accelerating factor (DAF), CR1, CR2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. Examples of such receptors includeflt-1, flk-1, TIE-2; the trk family of receptors such as TrkA, MuSK,Eph, PDGF receptor, EGF receptor, HER2, insulin receptor, IGF-1receptor, the FGF family of receptors, the TGFβ receptors, theinterleukin receptors, the interferon receptors, serotonin receptors,α-adrenergic receptors, β-adrenergic receptors, the GDNF receptor, p75neurotrophin receptor, among others. The invention encompasses receptorsfor extracellular matrix proteins, such as integrins, counter-receptorsfor transmembrane-bound proteins, such as intercellular adhesionmolecules (ICAM-1, ICAM-2, ICAM-3 and ICAM-4), vascular cell adhesionmolecules (VCAM), and selectins E-selectin, P-selectin and L-selectin.The invention encompasses receptors for cholesterol regulation,including the LDL receptor, HDL receptor, VLDL receptor, and thescavenger receptor. The invention encompasses the apolipoprotein ligandsfor these receptors, including ApoAI, ApoAIV and ApoE. The inventionalso encompasses gene products such as steroid hormone receptorsuperfamily including glucocorticoid receptors and estrogen receptors,Vitamin D receptors and other nuclear receptors. In addition, usefulgene products include antimicrobial peptides such as defensins andmaginins, transcription factors such as jun, fos, max, mad, serumresponse factor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, NF-κB,members of the leucine zipper family, C₂H₄ zinc finger proteins,including Zif268, EGR1, EGR2, C6 zinc finger proteins, including theglucocorticoid and estrogen receptors, POU domain proteins, exemplifiedby Pit 1, homeodomain proteins, including HOX-1, basic helix-loop-helixproteins, including myc, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZFS, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor 1 (IRF-1),Wilms' tumor protein, ETS-binding protein, STAT, GATA-box bindingproteins, e.g., GATA-3, and the forkhead family of winged helixproteins.

Other useful gene products include carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VII, factor VIII, factor IX, factor II, factor V, factor X,factor XII, factor XI, von Willebrand factor, superoxide dismutase,glutathione peroxidase and reductase, heme oxygenase, angiotensinconverting enzyme, endothelin-1, atrial natriuretic peptide,pro-urokinase, urokinase, plasminogen activator, heparin cofactor II,activated protein C (Factor V Leiden), Protein C, antithrombin,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase (also referred to asP-protein), H-protein, T-protein, Menkes disease protein, tumorsuppressors (e.g., p53), cystic fibrosis transmembrane regulator (CFTR),the product of Wilson's disease gene PWD, Cu/Zn superoxide dismutase,aromatic amino acid decarboxylase, tyrosine hydroxylase, acetylcholinesynthetase, prohormone convertases, protease inhibitors, lactase,lipase, trypsin, gastrointestinal enzymes including chymotrypsin, andpepsin, adenosine deaminase, α1 anti-trypsin, tissue inhibitor ofmetalloproteinases (TIMP), GLUT-1, GLUT-2, trehalose phosphate synthase,hexokinases I, II and III, glucokinase, any one or more of theindividual chains or types of collagen, elastin, fibronectin,thrombospondin, vitronectin and tenascin, and suicide genes such asthymidine kinase and cytosine deaminase. Other useful proteins includethose involved in lysosomal storage disorders, including acidβ-glucosidase, α-galactosidase a, α-1-iduronidase, iduroate sulfatase,lysosomal acid α-glucosidase, sphingomyelinase, hexosaminidase A,hexomimidases A and B, arylsulfatase A, acid lipase, acid ceramidase,galactosylceramidase, α-fucosidase, α-, β-mannosidosis,aspartylglucosaminidase, neuramidase, galactosylceramidase,heparan-N-sulfatase, N-acetyl-α-glucosaminidase, Acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatesulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B,β-glucuoronidase and hexosaminidases A and B.

Other useful transgenes include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides or polypeptides having anon-naturally occurring amino acid sequence containing insertions,deletions or amino acid substitutions. For example, single-chainengineered immunoglobulins could be useful in certain immunocompromisedpatients. Other useful proteins include truncated receptors which lacktheir transmembrane and cytoplasmic domain. These truncated receptorscan be used to antagonize the function of their respective ligands bybinding to them without concomitant signaling by the receptor. Othertypes of non-naturally occurring gene sequences include sense andantisense molecules and catalytic nucleic acids, such as ribozymes,which could be used to modulate expression of a gene.

Viral Vectors

Compositions as described herein (e.g., compositions including a viralvector encoding GAA) may be administered to a mammalian subject by anysuitable technique. Various techniques using viral vectors for theintroduction of a GAA gene into cells are provided for according to thecompositions and methods described herein. Viruses are naturally evolvedvehicles which efficiently deliver their genes into host cells andtherefore are desirable vector systems for the delivery of therapeuticgenes. Preferred viral vectors exhibit low toxicity to the host cell andproduce therapeutic quantities of GAA protein (e.g., in atissue-specific manner). Viral vector methods and protocols are reviewedin Kay et al., Nature Medicine, 7:33-40, 2001.

Although the experiments described below involve rAAV, any suitableviral vector can be used. Many viral vectors are known in the art fordelivery of genes to mammalian subject and a non-exhaustive list ofexamples follows. Methods for use of recombinant Adenoviruses as genetherapy vectors are discussed, for example, in W. C. Russell, J. Gen.Virol., 81:2573-2604, 2000; and Bramson et al., Curr. Opin. Biotechnol.,6:590-595, 1995. Methods for use of Herpes Simplex Virus vectors arediscussed, for example, in Cotter and Robertson, Curr. Opin. Mol. Ther.1:633-644, 1999. Replication-defective lentiviral vectors, includingHIV, may also be used. Methods for use of lentiviral vectors arediscussed, for example, in Vigna and Naldini, J. Gene Med., 5:308-316,2000 and Miyoshi et al., J. Virol., 72:8150-8157, 1998. Retroviralvectors, including Murine Leukemia Virus-based vectors, may also beused. Methods for use of retrovirus-based vectors are discussed, forexample, in Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong etal., Crit. Rev. Ther. Drug Carrier Syst., 17:1-60, 2000. Other viralvectors that may find use include Alphaviruses, including Semliki ForestVirus and Sindbis Virus. Hybrid viral vectors may be used to deliver agaa gene to a target tissue (e.g., muscle, central nervous system).Standard techniques for the construction of hybrid vectors arewell-known to those skilled in the art. Such techniques can be found,for example, in Sambrook et al., In Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y.), or any number of laboratory manualsthat discuss recombinant DNA technology.

rAAV Vectors and Virions

In some embodiments, nucleic acids of the compositions and methodsdescribed herein are incorporated into rAAV vectors and/or virions inorder to facilitate their introduction into a cell. rAAV vectors usefulin the invention are recombinant nucleic acid constructs that include(1) a heterologous sequence to be expressed (e.g., a polynucleotideencoding a GAA protein) and (2) viral sequences that facilitateintegration and expression of the heterologous genes. The viralsequences may include those sequences of AAV that are required in cisfor replication and packaging (e.g., functional ITRs) of the DNA into avirion. In typical applications, the heterologous gene encodes GAA,which is useful for correcting a GAA-deficiency in a cell. Such rAAVvectors may also contain marker or reporter genes. Useful rAAV vectorshave one or more of the AAV WT genes deleted in whole or in part, butretain functional flanking ITR sequences. The AAV ITRs may be of anyserotype (e.g., derived from serotype 2) suitable for a particularapplication. Methods for using rAAV vectors are discussed, for example,in Tal, J. Biomed. Sci., 7:279-291, 2000; and Monahan and Samulski, GeneDelivery, 7:24-30, 2000.

The nucleic acids and vectors of the invention are generallyincorporated into a rAAV virion in order to facilitate introduction ofthe nucleic acid or vector into a cell. The capsid proteins of AAVcompose the exterior, non-nucleic acid portion of the virion and areencoded by the AAV cap gene. The cap gene encodes three viral coatproteins, VP1, VP2 and VP3, which are required for virion assembly. Theconstruction of rAAV virions has been described. See, e.g., U.S. Pat.Nos. 5,173,414, 5,139,941, 5,863,541, and 5,869,305, 6,057,152,6,376,237; Rabinowitz et al., J. Virol., 76:791-801, 2002; and Bowles etal., J. Virol., 77:423-432, 2003.

rAAV virions useful in the invention include those derived from a numberof AAV serotypes including 1, 2, 3, 4, 5, 6, 7, 8 and 9. For targetingmuscle cells, rAAV virions that include at least one serotype 1 capsidprotein may be particularly useful as the experiments reported hereinshow they induce significantly higher cellular expression of GAA than dorAAV virions having only serotype 2 capsids. rAAV virions that includeat least one serotype 6 capsid protein may also be useful, as serotype 6capsid proteins are structurally similar to serotype I capsid proteins,and thus are expected to also result in high expression of GAA in musclecells. rAAV serotype 9 has also been found to be an efficient transducerof muscle cells. Construction and use of AAV vectors and AAV proteins ofdifferent serotypes are discussed in Chao et al., Mol. Ther. 2:619-623,2000; Davidson et al., Proc. Natl. Acad. Sci. USA, 97:3428-3432, 2000;Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol.74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; andAuricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001.

Also useful in the invention are pseudotyped rAAV. Pseudotyped vectorsof the invention include AAV vectors of a given serotype (e.g., AAV2)pseudotyped with a capsid gene derived from a serotype other than thegiven serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9 etc.). For example, a representative pseudotyped vector of theinvention is a rAAV2 vector encoding GAA pseudotyped with a capsid genederived from AAV of a different serotype (e.g., AAV1, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9). In one embodiment, it was observed that LacZtransgene delivery using the IV administration route and rAAV2/9pseudotype capsid results in approximately 200 fold higher levels ofexpression in cardiac tissue than an identical dose with rAAV2/1.Additional experiments indicated that IV delivery of a transgene usingrAAV2/9 to adult mice also results in transduction of cardiac tissue.Techniques involving the construction and use of pseudotyped rAAVvirions are known in the art and are described in Duan et al., J.Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532,2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio etal., Hum. Molec. Genet., 10:3075-3081, 2001.

AAV virions that have mutations within the virion capsid may be used toinfect particular cell types more effectively than non-mutated capsidvirions. For example, suitable AAV mutants may have ligand insertionmutations for the facilitation of targeting AAV to specific cell types.The construction and characterization of AAV capsid mutants includinginsertion mutants, alanine screening mutants, and epitope tag mutants isdescribed in Wu et al., J. Virol., 74:8635-45, 2000. Other rAAV virionsthat can be used in methods of the invention include those capsidhybrids that are generated by molecular breeding of viruses as well asby exon shuffling. See Soong et al., Nat. Genet., 25:436-439, 2000; andKolman and Stemmer, Nat. BiotechnoL, 19:423-428, 2001.

Modulating GAA Levels in a Cell

The nucleic acids, vectors, and virions described above can be used tomodulate levels of GAA in a cell. The method includes the step ofadministering to the cell a composition including a nucleic acid thatincludes a polynucleotide encoding GAA interposed between two AAV ITRs.The cell can be from any animal into which a nucleic acid of theinvention can be administered. Mammalian cells (e.g., human beings,dogs, cats, pigs, sheep, mice, rats, rabbits, cattle, goats, etc.) froma subject with GAA deficiency are typical target cells for use in theinvention.

In some embodiments, the cell is a myocardial cell, e.g., amyocardiocyte. In other embodiments, the cell is a neuron (e.g., phrenicmotor nerve).

Increasing Motoneuron (e.g., Phrenic Neuron) Function in a Mammal

rAAV vectors, compositions and methods described herein can be used toincrease phrenic nerve activity in a mammal having Pompe disease and/orinsufficient GAA levels. For example, rAAV encoding GAA can beadministered to the central nervous system (e.g, neurons). In anotherexample, retrograde transport of a rAAV vector encoding GAA from thediaphragm (or other muscle) to the phrenic nerve or other motor neuronscan result in biochemical and physiological correction of Pompe disease.These same principles could be applied to other neurodegenerativedisease.

Increasing GAA Activity in a Subject

The nucleic acids, vectors, and virions described above can be used tomodulate levels of functional GAA in an animal subject. The methodincludes the step of providing an animal subject and administering tothe animal subject a composition including a nucleic acid that includesa polynucleotide encoding GAA interposed between two AAV ITRs. Thesubject can be any animal into which a nucleic acid of the invention canbe administered. For example, mammals (e.g., human beings, dogs, cats,pigs, sheep, mice, rats, rabbits, cattle, goats, etc.) are suitablesubjects. The methods and compositions of the invention are particularlyapplicable to GAA-deficient animal subjects.

The compositions described above may be administered to animalsincluding human beings in any suitable formulation by any suitablemethod. For example, rAAV virions (i.e., particles) may be directlyintroduced into an animal, including by intravenous (IV) injection,intraperitoneal (IP) injection, or in situ injection into target tissue(e.g., muscle). For example, a conventional syringe and needle can beused to inject a rAAV virion suspension into an animal. Depending on thedesired route of administration, injection can be in situ (i.e., to aparticular tissue or location on a tissue), IM, IV, IP, or by anotherparenteral route. Parenteral administration of virions by injection canbe performed, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the rAAV virions may be in powder form (e.g.,lyophilized) for constitution with a suitable vehicle, for example,sterile pyrogen-free water, before use.

To facilitate delivery of the rAAV virions to an animal, the virions ofthe invention can be mixed with a carrier or excipient. Carriers andexcipients that might be used include saline (especially sterilized,pyrogen-free saline) saline buffers (for example, citrate buffer,phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids,urea, alcohols, ascorbic acid, phospholipids, proteins (for example,serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,and glycerol. USP grade carriers and excipients are particularly usefulfor delivery of virions to human subjects. Methods for making suchformulations are well known and can be found in, for example,Remington's Pharmaceutical Sciences.

In addition to the formulations described previously, the virions canalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by IM injection. Thus, for example, the virions maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives.

Similarly, rAAV vectors may be administered to an animal subject using avariety of methods. rAAV vectors may be directly introduced into ananimal by intraperitoneal administration (IP injection), as well asparenteral administration (e.g., IV injection, IM injection, and in situinjection into target tissue). Methods and formulations for parenteraladministration described above for rAAV virions may be used toadminister rAAV vectors.

Ex vivo delivery of cells transduced with rAAV virions is also providedfor within the invention. Ex vivo gene delivery may be used totransplant rAAV-transduced host cells back into the host. Similarly, exvivo stem cell (e.g., mesenchymal stem cell) therapy may be used totransplant rAAV vector-transduced host cells back into the host. Asuitable ex vivo protocol may include several steps. A segment of targettissue (e.g., muscle, liver tissue) may be harvested from the host andrAAV virions may be used to transduce a GAA-encoding nucleic acid intothe host's cells. These genetically modified cells may then betransplanted back into the host. Several approaches may be used for thereintroduction of cells into the host, including intravenous injection,intraperitoneal injection, or in situ injection into target tissue.Microencapsulation of cells transduced or infected with rAAV modified exvivo is another technique that may be used within the invention.Autologous and allogeneic cell transplantation may be used according tothe invention.

Effective Doses

The compositions described above are typically administered to a mammalin an effective amount, that is, an amount capable of producing adesirable result in a treated subject (e.g., increasing WT GAA activityin the subject). Such a therapeutically effective amount can bedetermined as described below.

Toxicity and therapeutic efficacy of the compositions utilized inmethods of the invention can be determined by standard pharmaceuticalprocedures, using either cells in culture or experimental animals todetermine the LD₅₀ (the dose lethal to 50% of the population). The doseratio between toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio LD₅₀/ED₅₀. Those compositions thatexhibit large therapeutic indices are preferred. While those thatexhibit toxic side effects may be used, care should be taken to design adelivery system that minimizes the potential damage of such sideeffects. The dosage of compositions as described herein lies generallywithin a range that includes an ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

As is well known in the medical and veterinary arts, dosage for any oneanimal depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, timeand route of administration, general health, and other drugs beingadministered concurrently. It is expected that an appropriate dosage forintravenous administration of particles would be in the range of about10¹²-10¹⁵ particles. For a 70-kg human, a 1- to 10-mL (e.g., 5 mL)injection of 10¹²-10¹⁵ particles is presently believed to be anappropriate dose.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Materials and Methods Virus Production

Recombinant AAV vectors were generated, purified, and titered at theUniversity of Florida Powell Gene Therapy Center Vector Core Laboratoryas previously described (Zolotukhin, S, et al., Methods, 28:158-167,2002).

Intravenous Injections

All animal procedures were performed in accordance with the Universityof Florida Institutional Animal Care and Use Committee (IACUC)guidelines (mice) or the University of California (UC) Davis IACUC(monkeys; see below). One-day-old mouse pups were injected via thesuperficial temporal vein as previously described (Sands M S, et al.,Lab. Anim. Sci., 49:328-330, 1999). Briefly, mice were anesthetized byinduced hypothermia. A 29.5-gauge tuberculin syringe was used to delivervector in a total volume of 35 μL directly into the left temporal vein.Two-month-old adult mice were injected via the jugular vein. Mice werefirst anesthetized using a mixture of 1.5% isoflurane and O₂ (1 to 2 L).A 0.5-cm incision was made to expose the jugular vein. A 29-gaugesterile needle and syringe were then used to deliver virus in a volumeof 150 Hemostasis was obtained; the skin was approximated and heldsecure with Vetbond (3M, St. Paul, Minn.).

β-Galactosidase Detection

Tissue lysates were assayed for β-galactosidase enzyme activity usingthe Galacto-Star chemiluminescence reporter gene assay system (TropixInc, Bedford, Mass.). Protein concentrations for tissue lysates weredetermined using the Bio-Rad DC protein assay kit (Hercules, Calif.).

ECG Analysis

ECG tracings were acquired using standard subcutaneous needle electrodes(MLA1203, 1.5 mm Pin 5; AD Instruments) in the right shoulder, rightforelimb, left forelimb, left hindlimb, and tail and a Power LaboratoryDual BioAmp instrument. Five minutes of ECG tracings from each animalwere analyzed using ADInstrument's Chart® software.

Nonhuman Primate Studies

Studies with monkeys were conducted in the Center for Fetal Monkey GeneTransfer for Heart, Lung, and Blood Diseases located at the CaliforniaNational Primate Research Center (UC Davis). Gravid rhesus monkeys (n=6)were monitored during pregnancy by ultrasound, and newborns delivered bycesarean section at term using established techniques. Within an hour ofbirth, newborns were injected intravenous with vector mL) via aperipheral vessel. Infants received either rAAV2/1-CMV-hGaa (n=3) orrAAV2/9-CMV-hGaa (n=3). Infants were nursery reared and monitored for 6months and then euthanized by an overdose of pentobarbital and completetissue harvests performed (one per group) using established methods.Specimens from control animals of a comparable age were made availablethrough the Center for Fetal Monkey Gene Transfer. GAA activity wasmeasured from tissues harvested 6 months postinjection and backgroundactivity from non-injected controls was subtracted to yield the resultsin FIG. 5A. Genomic DNA (gDNA) was extracted from tissues according tothe protocol of the manufacturer (Qiagen; DNeasy tissue kit). ResultingDNA concentrations from the extraction procedure were determined usingan Eppendorf Biophotometer (Model 6131; Eppendorf, Hamburg, Germany).One microgram of extracted gDNA was used in all quantitative PCRsaccording to a previously used protocol (Song, S, et al., Mol Ther.,6:329-335, 2002) and reaction conditions (recommended byPerkin-Elmer/Applied Biosystems) included 50 cycles of 94.8° C. for 40seconds, 37.8° C. for 2 minutes, 55.8° C. for 4 minutes, and 68.8° C.for 30 seconds. Primer pairs were designed to the CMV promoter asdescribed (Donsante A, et al., Gene Ther., 8:1343-1346, 2001) andstandard curves established by spike-in concentrations of a plasmid DNAcontaining the same promoter. DNA samples were assayed in triplicate.The third replicate was supplemented with CBATDNA at a ratio of 100copies/μg of gDNA. If at least 40 copies of the spike-in DNA weredetected, the DNA sample was considered acceptable for reporting vectorDNA copies.

EXAMPLE 1 Recombinant Adeno-Associated Virus Leads to PreferentialCardiac Transduction In Vivo

rAAV2/1 was directly compared with two less-characterized serotypes(rAAV2/8 and rAAV2/9) in their abilities to transduce myocardium invivo. These recombinant or pseudotyped vectors are created by insertinga transgene of interest flanked by the inverted terminal repeats (ITRs)of AAV2 into the capsid of another serotype. 1×10¹¹ vector genomes (vg)were delivered of each of 3 different serotypes (rAAV2/1, rAAV2/8, orrAAV2/9) carrying the CMV-lacZ construct (cytoplasmic lacZ) by thesystemic venous route to 1-day-old mice (5 neonates per group) in aninjection volume of 35 μL (FIG. 1A-FIG. 1D). Hearts from the injectedmice were harvested at 4 weeks postinjection and5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) staining wasperformed on frozen cryosections to visualize the extent ofβ-galactosidase expression biodistribution across the myocardium (FIG.1A-FIG. 1C). In addition, β-galactosidase activity was determined toquantify LacZ expression (FIG. 1D). The SYBR green quantitative PCRtechnique was also performed on these hearts to compare the relativeamounts of vector genomes present. It was found that 0.19, 16.12, and76.95 vg per diploid cell were present in the hearts of mice injectedwith rAAV2/1, rAAV2/8, and rAAV2/9, respectively.

Calculations for vector genomes per cell were determined as previouslydescribed (Wei J F, et al., Gene Ther., 1:261-268, 1994).

The results show that of those serotypes compared in this work, systemicvenous delivery of AAV2/9 results in broad and even distribution ofvector and transgene product in the myocardium without selective cardiacadministration. The level of gene expression was shown to result in a200-fold greater level of expression than that observed for rAAV2/1.rAAV2/8 provides exceptional transduction of myocardium at levels≈20-fold greater than those obtained using rAAV2/1; however, there isalso significant transduction of hepatocytes with this serotype.X-Gal-stained cryosections demonstrated that both rAAV2/8 and rAAV2/9provide a broad and even distribution of transgene expression throughoutthe entire heart. In contrast, those hearts injected with rAAV2/1 showedfar less overall expression. Additionally, immunohistochemistry with acardiac troponin antibody (Santa Cruz Biotechnology) was performed andit was found that the cells expressing β-galactosidase werecardiomyocytes.

Also, studies demonstrated that rAAV2/9 transduced cardiomyocytes moreefficiently than myoblasts in vitro. The β-galactosidase enzymedetection assay was then performed on other tissues from these sameanimals to characterize the biodistribution of lacZ expression. It wasfound that rAAV2/8 and rAAV2/9 are both capable of transduction ofskeletal muscle to some degree (FIG. 2A). In general, rAAV2/8 has theability to provide an overall broad and even biodistribution ofexpression across muscle in addition to the heart, whereasrAAV2/9-delivered transgene expression is far greater in the heart thanany other tissue.

The β-galactosidase assay was also performed on noncardiac, nonskeletalmuscle tissue samples from these mice (FIG. 2B). These results showedthat whereas rAAV2/8 and rAAV2/9 are able to transduce tissues such asbrain, lung and kidney, there is less transduction of spleen and smallintestine.

Once it was established that rAAV2/9 displayed the highest naturalaffinity for myocardium, rAAV2/9 activity was further characterized invivo. The CMV promoter was chosen for these studies because thisexpression cassette was appropriate in size and expression profile inthe target tissue of interest. SYBR green quantitative PCR was performedon heart, liver, and quadriceps tissue specimens from mice that wereinjected with rAAV2/9 to compare the relative amounts of vector genomespresent in these tissues. These results showed that there were ≈76.95vg/cell (vector genomes per diploid cell) in myocardium and 2.89 vg/celland 11.47 vg/cell present in liver and quadriceps, respectively. Theclinical implication of these findings is that even when using an AAVcapsid, which displays a high natural affinity for a specific tissue,the use of a tissue-specific promoter will be critical to ultimatelyensure restricted transgene expression to the area of interest.

Additional studies were performed to evaluate a time course assay ofrAAV2/9-CMV-lacZ expression in cardiac and skeletal muscle. One-day-oldmouse neonates were injected with 5×10¹⁰ vg, and cardiac and skeletalmuscles were harvested at 1, 7, 14, 28, and 56 days postinjection (FIG.3A). The results show that the onset of transgene expression in bothtissues occurred between 1 and 7 days following administration ofvector. The amount of expression in skeletal muscle increased graduallyover the first 28 days, then leveled off and sustained a constant levelout to at least 56 days. The amount of transgene expression in cardiactissue was consistently higher than that in skeletal muscle andcontinued to steadily increase throughout the duration of the experiment(56 days).

SYBR green quantitative PCR was next performed on these tissues todetermine whether the increase of transgene expression in cardiac tissuewas attributable to an increase in β-galactosidase protein stability incardiac tissue as compared with skeletal muscle tissue (FIG. 3B). Vectorgenome copy number increased in cardiac tissue but not skeletal muscletissue. Additionally, RNA was isolated from these tissues and it wasfound that RNA transcript numbers also increased over the duration ofthe experiment (FIG. 3C).

The cellular receptor for AAV9 is not currently known; however, thepreferential cardiac transduction warrants further evaluation of cardiacligands, which are bound by AAV. The data suggest that the AAV9 capsidmay not be absorbed by other tissues as easily as previously studiedserotypes because of its inability to bind to a more ubiquitous receptorlocated throughout the body, such as the heparin sulfate proteoglycanreceptor. Therefore the AAV9 capsid could require more time to reachcardiac tissue. An additional explanation for the increase in vectorgenome concentration over the course of the experiment is that there maybe a delay in the double-strand synthesis of the delivered transgene inthe heart that could potentially account for a doubling of vectorgenomes.

Next, a study in adult mice was performed to determine whether therAAV2/9 behavior that was observed in neonates is similar in adultanimals. rAAV2/9-CMV-lacZ (1×10¹¹ vg) was administered to 3-month-oldmice using an intravenous delivery route via the jugular vein (FIG. 4B).Tissues were harvested at 4 weeks postinjection, and the level oftransgene expression was determined for both cardiac and skeletalmuscle. The results show that rAAV2/9 does transduce cardiac andskeletal muscle in adult mice, although in comparison with the same doseadministered to neonates, the expression levels were far lower (FIG.4A). The rAAV2/9-delivered expression level in adults was comparable tothat observed following intravenous delivery of the same dose ofrAAV2/1-CMV-lacZ to neonates. Lower overall rAAV2/9 transduction inadults in comparison to the same dose in neonates is not unexpectedbecause of the reduced dose per kilogram of body weight. These datademonstrate, however, that a similar biodistribution profile is observedwhether rAAV2/9 is intravenously delivered to adults or neonates andprovide further evidence that rAAV2/9 preferentially transduces cardiactissue.

A model of inherited cardiomyopathy was used to assess a gene-transferapproach to this condition. Pompe disease is a form of musculardystrophy and metabolic myopathy caused by mutations in the acidβ-glucosidase (Gaa) gene. An insufficient amount of the GAA enzyme leadsto the accumulation of glycogen in lysosomes and consequent cellulardysfunction. In human patients, there is a direct correlation betweenthe amount of GAA produced and the severity of disease. Withouttreatment, cardiorespiratory failure typically occurs in the early-onsetpatients within the first year of life.

To demonstrate the ability of the rAAV2/9 pseudotype to deliver atherapeutic transgene to correct and/or prevent the onset of a diseasephenotype, the Gaa^(−/−) mouse model was treated with rAAV2/9-CMV-hGaa(human Gaa). Because of the rAAV2/9 marker gene results, it wasanticipated that a lower therapeutic dose than is typically necessarywould be sufficient to provide correction in a mouse model ofcardiomyopathy. Therefore, doses per neonate of either 4×10⁵ or 4×10⁸ vgof rAAV2/9-CMV-hGaa were administered to Gaa^(−/−) mice at 1 day of ageusing the intravenous delivery route. At 3 months postinjection, ECGswere performed on each dosage group of treated mice and noninjected, agematched Gaa^(−/−) and healthy wild-type (B6/129) controls.

Similar to the human form of this disease, untreated Gaa^(−/−) mouseECGs display a shortened PR interval as compared with healthy B6/129controls (PR=33.41±1.35 ms, or 26% shorter than wild type [PR=44.95±1.58ms]). The mice that were treated with the low dose (4×10⁵ vg) ofrAAV2/9-CMV-hGaa displayed a PR interval of 36.76±1.12 ms, or only 18%shorter than wild-type, age-matched controls (P=0.062). The dosage grouptreated with 4×10⁸ vg displayed a PR interval of 39.38±2.42 ms, or only12% shorter than B6/129 age-matched controls (P=0.058). Essentially, atthese low doses, a lengthened PR interval was observed that may increaseas time progresses.

Although the mouse is a generally well-accepted model for gene therapystudies, behavior of the various AAV capsids in humans may be quitedifferent. Therefore, long-term experiments are presently beingperformed in nonhuman primates to assess the expression over time in ananimal model more phylogenetically similar to humans. Results from thisongoing study show that at 6 months following intravenous delivery via aperipheral vessel (at birth) of rAAV2/9-CMV-hGaa or rAAV2/1-CMV-hGaa toinfant rhesus macaques, the expression profile between serotypes issimilar to what was observed in mice with rAAV2/9, providing 4-fold moreGAA expression than rAAV2/1 (FIG. 5A). The vector genome biodistributionprofile observed in these nonhuman primate tissues was also similar towhat was found in mouse tissue (FIG. 5B) with rAAV2/9, demonstrating adramatic preference for cardiac tissue over skeletal muscle. For boththe expression and vector genome analysis of nonhuman primate heartspecimens, numbers were averaged between right and left heart includingthe atria and ventricles. Biodistribution of expression and vectorgenomes appeared to be even throughout the heart.

EXAMPLE 2 The AAV9 Capsid Preferentially Transduces Cardiac Tissue andDemonstrates Unique Behavior In Vivo Development of a Gene TherapyApproach for the Treatment of Inherited Cardiomyopathies:

By assessing expression profiles in tissues throughout the bodyfollowing intravenous (IV) administration of virus to adults, newbornmice and non-human primates, it was determined that (of those assessed)the optimal AAV serotype for transduction of cardiac tissue is AAV2/9.Through MRI, ECG and tissue analysis, it was demonstrated that IVdelivery of 4¹⁰ vg AAV2/9 carrying a therapeutic transgene canameliorate the cardiac phenotype in a mouse model of Pompe disease, aglycogen storage disorder. At 3 month of age ECG analysis showedimprovement in PR interval and MRI assessment demonstrated increasedcardiac output as compared to untreated controls At 6 months postadministration these improvements continued and PAS stains of heartspecimens showed successful clearance of glycogen. The high naturalaffinity of AAV2/9 for cardiac tissue suggests that it preferentiallybinds a receptor that is prevalent in cardiomyocytes. The studies haveunveiled an interesting feature that is unique to this capsid amongthose previously worked with. 5¹⁰ vg of AAV2/9-CMV-LacZ wereadministered to 1 day old mice and heart and muscles were harvested in atime course out to 56 days to quantify expression. While beta-galexpression leveled off in skeletal muscle tissue, it continued toincrease in heart. Analysis of vg revealed the same phenomenon. The datasuggests that AAV9 capsids may continue to be released from tissues overtime and require more time to reach the heart following IV delivery.

rAAV2/9 Mediated Gene Delivery of Acid α-Glucosidase Corrects theCardiac Phenotype in a Mouse Model of Pompe Disease:

Pompe Disease is a form of muscular dystrophy and metabolic myopathycaused by mutations in the acid alpha glucosidase (GAA) gene. Aninsufficient amount of GAA leads to the accumulation of glycogen inlysosomes and consequent cellular dysfunction. In human patients thereis a direct correlation between the amount of GAA produced and severityof disease. Without treatment, cardio-respiratory failure typicallyoccurs in the early onset patients within the first year of life.

Described herein is a characterization study of the cardiac phenotype inthe GAA knockout mouse model (gaa−/−) at various ages through analysisof ECG traces, MRI data and use of the periodic acid shift (PAS) stainto visually assess glycogen content in tissue sections. Through ECGanalysis, a shortened PR interval was observed by 3 months of age(gaa−/− 33.41±1.35 ms, control 44.95±1.58 ms) mimicking the conductionphenotype observed in the human Pompe population. By 2 weeks of ageabnormal amounts of glycogen can be observed in the lysosomes of cardiaccells as demonstrated by the PAS stain. MRI analysis shows a decrease instroke volume (SV) (gaa−/− 36.13±1.19 μL, control 51.84±3.59 μL) and adecrease in cardiac output (CO) (gaa−/− 7.95±0.26 mL/min, control11.40±0.79 mL /min) at 3 months and a significant increase in myocardialmass (gaa−/− 181.99±10.7 mg, control 140.79±5.12 mg) by 12 months ofage.

This model of cardiac dysfunction is used in order to develop a cardiacgene delivery technique which can be applied to many geneticallyinherited cardiomyopathies. It was previously shown that IV delivery ofrecombinant AAV2 viral vectors pseudotyped with viral capsids ofserotype 1 (rAAV2/1) carrying the CMV-hGAA construct to 1 day old Gaa−/−neonates restores GAA activity in various tissues when observed 12months post-administration. More recently, it was found that LacZtransgene delivery using the IV administration route and rAAV2/9pseudotype capsid results in approximately 200 fold higher levels ofexpression in cardiac tissue than an identical dose with rAAV2/1.Additional experiments indicated that IV delivery of a transgene usingrAAV2/9 to adult mice also results in transduction of cardiac tissue.

The most optimal rAAV serotype for cardiac transduction (rAAV2/9) hasnow been combined with the clinically relevant IV administration routein order to deliver the human GAA (hGAA) gene to Gaa−/− mice. Neonatestreated with rAAV2/9-CMV-hGAA at a range of doses (4×10⁵ vg, 4×10⁸ vgand 4×10¹⁰ vg) using this strategy have demonstrated sustainedcorrection as assessed by ECG analysis (39.38±2.42 ms). PAS stains onfrozen tissue sections as well as NMR analysis on lyophilized tissueshave shown less glycogen accumulation in cardiac tissue of gaa−/− micetreated as neonates as compared to untreated controls. Non-invasive MRIanalysis has shown an increase in SV and CO. Adult Gaa−/− mice have alsobeen treated using the IV delivery route and are currently beingassessed in order to reverse the effects of Pompe Disease in mice whichhave already begun presenting the cardiac phenotype.

The systemic delivery route, use of the CMV promoter and the fact thatGAA is a secreted enzyme all promote expression and correctionthroughout the body. GAA activity has been observed in various othertissues of treated mice including skeletal muscles and liver. Inconclusion, these studies have demonstrated the ability of rAAV2/9 to beadministered systemically using a relatively noninvasive IV deliveryroute, transcend the vasculature, transduce tissues throughout the bodyand ultimately prevent presentation of the cardiac phenotypes of PompeDisease.

EXAMPLE 3 MRI for Characterization and Gene Therapy Evaluation in MurineModels of Muscular Dystrophy

Studies to establish which combination of adeno-associated virus (AAV)serotype, promoter and delivery route is the most advantageous forcardiac gene delivery are performed. Studies to non-invasivelycharacterize hearts in mouse models of the various forms of musculardystrophy which can be treated are performed. Examples of models ofvarious forms of muscular dystrophy include: a model for Limb GirdleMuscular Dystrophy; alpha-sarcoglycan knockout (ASG−/−), a model forMyotonic Dystrophy Type 1 (MDNL1−/−) in which exon 3 of MBNL has beendeleted and the MDX mouse model for Duchenne Muscular Dystrophy whichlacks dystrophin.

In initial characterization studies, cardiac tissue from these modelswas harvested at a range of ages and found that the manifestations ofdisease increase with age in all cases. The location and size ofdystrophic lesions in the early stages of development can be identifiedand determined because of their ability to uptake and sequester thefluorescent dye, Evans Blue Dye (EBD), due to the abnormal permeabilityof deteriorated muscle tissue. The ability to non-invasively identifyand monitor the progression of dystrophic lesion development in skeletalmuscle using ¹H-magnetic resonance techniques was also demonstrated. Inorder to recognize lesions in the later stages of development oncryosections, the trichrome stain was utilized. This stains for thepresence of collagen that infiltrates more progressed dystrophic lesionsas they undergo fibrosis.

Cardiac MR provides high-resolution images that offer structural as wellas global and regional functional information. In older MDX mice (6-52wk), the heart shows focal lesions of inflammatory cell infiltration,myocyte damage and fibrosis generally located in the ventricle orseptum. It was also found that the older MDX hearts (>48 wks) displayregions of increased MR signal intensity. The hyper intense regionscorrelated with regions of myocyte damage, as determined histologicallyusing EBD accumulation, H&E, and trichrome staining. Cardiac MR can alsobe used to monitor myocyte function. By performing cardiac MRI on thesemodels at various ages, images were obtained which have enabled theidentification of the presentation of dilated cardiomyopathy,contractility defects and arrhythmias. In addition to standard cardiacimaging measurements and techniques, cardiac tagging protocols are beingestablished to allow the identification of areas of localizedcontractility defects. This may be beneficial for mouse models which maydisplay regional dysfunction due to areas of necrotic tissue throughoutthe heart.

Upon completion of these characterization studies, a next step includesproviding gene therapy to these mice and prevention of themanifestations of these diseases. The treated animals are thenperiodically non-invasively assessed using established MRI protocols inorder to ultimately demonstrate functional correction in murine modelsof cardiomyopathy.

EXAMPLE 4 Neural Deficits Contribute to Respiratory Insufficiency toPompe Disease

The main objectives were to determine if GAA^(−/−) mice have an alteredpattern of breathing, similar to the ventilation difficulties observedin the patient population and whether ventilation deficits in GSD II aremediated by a central component.

Plethysmography: Barometric plethysmography was used to measure minuteventilation (MV) and inspiratory time (T_(i)) in GAA^(−/−) mice andage-matched controls (B6/129 strain). After an acclimation period (30min) and baseline (60 min; F_(i)O₂=21%, F_(i)O₂=0%), mice were exposedto hypercapnic challenge (10 min, F_(i)CO₂=6.5%) to stimulaterespiratory motor output.

Blood Sampling: Control (B6/129) and GAA−/− mice were anesthetized and˜100 μL tail blood collected into a disposable G8+ cartridge and readwith a portable I-Stat machine (Heska Corp.).

Glycogen Detection: Glycogen was quantified using a modification of theacid-hydrolysis method. Periodic Acid Schiff stain was performed forhistological glycogen detection; Fluoro-Gold® (4%) was painted ontomouse diaphragms 48 hours prior to sacrifice for detection of phrenicmotoneurons.

Force Frequency Measurements in vitro: The optimal length for isometrictetanic tension was determined for each diaphragm strip followed byprogressively increasing stimulation frequency. Force generated wasnormalized to diaphragm strip length and weight.

Neurophysiology: The right phrenic nerve was isolated and electricalactivity recorded in anesthetized (urethane, i.v. 1.0-1.6 g/kg),mechanically ventilated, paralyzed and vagotomized mice with a bipolartungsten electrode.

Results and Summary: FIG. 6A-FIG. 6C are graphs showing the results ofminute ventilation (mL/min) at baseline and during 10 minutes ofhypercapnia in 6 month (FIG. 6A), 12 month (FIG. 6B) and >21 month (FIG.6C) control and GAA^(−/−) mice.

In summary, the results show:

GAA^(−/−) mice have an altered pattern of breathing compared to agematched control mice (FIG. 7A).

GAA deficiency in the nervous system results in ventilation deficits asdemonstrated by attenuated minute ventilation in muscle specific GAAmice (which have normal functioning diaphragm) (FIG. 7B).

The attenuated mean inspiratory flow suggests the drive to breathe inGAA^(−/−) mice may be decreased (FIG. 8).

Accumulation of glycogen in the spinal cord of GAA^(−/−) mice isobserved beginning at 6 months of age (FIG. 9A and FIG. 9B).

Efferent inspiratory phrenic output is reduced in GAA^(−/−) vs. control(FIG. 10A and FIG. 10B).

Conclusion: The ventilation deficits in GAA^(−/−) mice are similar tothe patient population. Mean inspiratory flow, glycogen quantification,muscle specific GAA mouse pattern of breathing and phrenic neurogramdata are consistent with the hypothesis that these ventilatorydifficulties reflect both a muscle and a neural component in GSD II.

EXAMPLE 5 Physiological Correction Of Pompe Disease Using RAAV2/1Vectors Materials and Methods

The recombinant AAV2 plasmid p43.2-GAA (Fraites, T. J., Jr. et al., Mol.Ther. 5:571-578, 2002) has been described previously. Recombinant AAVparticles based on serotype 1 were produced using p43.2-GAA and weregenerated, purified, and titered at the University of Florida PowellGene Therapy Center Vector Core Lab as previously described (Zolotukhin,S. et al., Methods, 28:158-167, 2002).

All animal studies were performed in accordance with the guidelines ofthe University of Florida Institutional Animal Care and Use Committee.The mouse model of Pompe disease (Gaa^(−/−)) used in this study has beendescribed previously and was generated by a targeted disruption of exon6 of the Gaa gene (Raben, N. et al., J. Biol. Chem., 273:19086-19092,1998). One-day-old Gaa^(−/−) mice were administered 5×10¹⁰ particles (30μL total volume) rAAV2/1-CMV-GAA intravenously via the superficialtemporal vein as described previously (Sands, M. S. and Barker, J. E.,Lab. Anim. Sci., 49:328-330, 1999).

Ten-, 24-, and 52-weeks' post-injection, tissue homogenates were assayedfor GAA enzyme activity. Briefly, lysates were assayed for GAA activityby measuring the cleavage of the synthetic substrate4-methylumbelliferyl-α-D-glucoside (Sigma M9766, Sigma-Aldrich, St.Louis, Mo.) after incubation for 1 h at 37° C. Successful cleavageyielded a fluorescent product that emits at 448 nm, as measured with anFLx800 microplate fluorescence reader (Bio-Tek Instruments, Winooski,Vt.). Protein concentration was measured using the Bio-Rad DC proteinassay kit (Bio-Rad, Hercules, Calif.). Data are represented aspercentage of normal levels of GAA in each tissue after subtraction ofuntreated Gaa^(−/−) tissue levels. Detection of anti-GAA antibodies wasperformed by ELISA.

Segments of treated and untreated diaphragm were fixed overnight in 2%glutaraldehyde in PBS, embedded in Epon 812® (Shell), sectioned, andstained with periodic acid-Schiff (PAS) by standard methods.

Mice were anesthetized with a mixture of 1.5-2% isoflurane and 1 L/minoxygen then positioned supine on a heating pad. ECG leads were placedsubcutaneously in the right shoulder, right forelimb, left forelimb,left hind limb and the tail. ECG tracings were acquired for five minutesper animal using PowerLab ADInstruments unit and Chart acquisitionsoftware (ADInstruments, Inc., Colorado Springs, Colo.). Peak intervalsfrom all tracings were averaged for each animal and then averaged withineach experimental group.

Assessment of Cardiac Mass

Cardiac MRI was performed on a 4.7 T Bruker Avance spectrometer (BrukerBioSpin Corporation, Billerica, Mass.) at the University of FloridaAdvanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) facility.The animals were anesthetized using 1.5% isoflurane (AbbottLaboratories, North Chicago, Ill.) and 1 L/min oxygen. The animals wereplaced prone on a home-built quadrature transmit-and-receive surfacecoil with the heart placed as near to the center of the coil aspossible. The images were acquired using cardiac gating and weretriggered at the peak of the R-R wave (SA Instruments, Inc., StonyBrook, N.Y.). The heart was visualized by acquiring single short axisslices along the length of the left ventricle. The images were acquiredusing a gradient recalled echo (GRE) sequence (matrix=256×128, TE=2.4ms, FOV=4 cm×3 cm, 7-8 slices, thickness=1 mm). The effective TR (pulserepetition time) was governed by the heart rate of the animal, which wasobserved to maintain consistency and anesthesia was adjustedaccordingly. The R-R interval was typically 250 ms.

Images were processed using CAAS MRV for mice (Pie Medical Imaging,Maastricht, The Netherlands). Contours were drawn for the epicardium andthe endocardium for each slice along the length of the left ventricle atboth end diastole and end systole. The results were exported andanalyzed and end diastolic myocardial mass was calculated.

Isometric force-frequency relationships were used to assess diaphragmcontractile force. The diaphragm is isolated, with the ribs and centraltendon attached, and placed in Krebs-Henseleit solution equilibratedwith a 95% O₂/5% CO₂ gas mixture on ice. A single muscle strip, cut fromthe ventral costal diaphragm parallel to the connective tissue fibers,is used to determine force-frequency relationships. Plexiglas® clampsare attached to the diaphragm strip via clamping to the rib and centraltendon. The muscle strip is suspended vertically in a water jacketedtissue bath (Radnoti, Monrovia, Calif.) containing Krebs-Henseleitsolution equilibrated with a 95% O₂/5% CO₂ gas mixture, maintained at37° C., pH 7.4, and equilibrated for 15 min. To measure isometriccontractile properties, the clamp attached to the central tendon isconnected to a force transducer (Model FT03, Grass Instruments, WestWarwick, R.I.). The transducer outputs are amplified and differentiatedby operational amplifiers and undergo A/D conversion using acomputer-based data acquisition system (Polyview, Grass Instruments). Todetermine the muscle strip optimal length (L_(o)) for isometric tetanictension, the muscle is field-stimulated (Model S48, Grass Instruments)along its entire length using platinum wire electrodes. Single twitchcontractions are evoked, followed by step-wise increases in musclelength, until maximal isometric twitch tension is obtained. Allcontractile properties are measured isometrically at L_(o). Peakisometric tetanic force is measured at 10, 20, 40, 80, 100, 150, and 200Hz. Single 500 ms trains are used, with a four-minute recovery periodbetween trains to prevent fatigue. Calipers are used to measure L_(o)before removal of the muscle from the apparatus. The muscle tissue isthen dissected away from the rib and central tendon, blotted dry, andweighed. The muscle cross-sectional area (CSA) is determined using theequation CSA (cm²)=[muscle strip mass (g)/fiber length L_(o) (cm)×1.056(g/cm³)], where 1.056 g/cm³ is the assumed density of muscle. Thecalculated CSA is used to normalize isometric tension, which isexpressed as N/cm².

Respiratory function was assayed using barometric whole bodyplethysmography. Unanesthetized, unrestrained C57BL6/129SvJ, Gaa^(−/−),and rAAV2/1-treated Gaa^(−/−) mice were placed in a clear Plexiglas®chamber (Buxco, Inc., Wilmington, N.C.). Chamber airflow, pressure,temperature, and humidity are continuously monitored and parameters suchas frequency, minute ventilation, tidal volume, and peak inspiratoryflow are measured and analyzed using the method by Drorbaugh and Fennand recorded using BioSystem XA software (Buxco, Inc.) (Drorbaugh, J. E.and Fenn, W. O., Pediatrics, 16:81-87, 1955). Baseline measurements aretaken under conditions of normoxia (F₁O₂: 0.21, F₁CO₂: 0.00) for aperiod of one hour followed by a ten minute exposure to hypercapnia(F₁O₂: 0.21, F₁CO₂: 0.07).

Results:

Cardiac and respiratory function in rAAV2/1-treated animals wasexamined. Similar to the Pompe patient population, electrocardiogram(ECG) measurements (P-R interval) were significantly shortened in themouse model. In rAAV2/1-treated mice, a significant improvement incardiac conductance with prolonged P-R intervals of 39.34±1.6 ms wasshown, as compared to untreated controls (35.58±0.57 ms) (p≦0.05). Inaddition, using cardiac magnetic resonance imaging (MRI), a markeddecrease in cardiac left ventricular mass was noted from 181.99±10.70 mgin untreated age-matched controls to 141.97±19.15 mg in therAAV2/1-treated mice. Furthermore, the mice displayed increaseddiaphragmatic contractile force to approximately 90% of wild-type peakforces with corresponding significantly improved ventilation(particularly in frequency, minute ventilation, and peak inspiratoryflow), as measured using barometric whole body plethysmography. Theseresults demonstrate that in addition to biochemical and histologicalcorrection, rAAV2/1 vectors can mediate sustained physiologicalcorrection of both cardiac and respiratory function in a model of fatalcardiomyopathy and muscular dystrophy.

Systemic Delivery of rAAV2/1 Can Result in Sustained Restoration ofCardiac and Diaphragmatic GAA Enzymatic Activity in Gaa^(−/−) Mice

5×10¹⁰ particles of rAAV2/1-CMV-hGAA were injected into one-day-oldGaa^(−/−) mice via the superficial temporal vein. Serial serum sampleswere collected to assay for the formation of anti-hGAA antibodies andcardiac and diaphragm tissues were analyzed for GAA enzyme activity atten, 24, and 52 weeks post-injection. A transient humoral immuneresponse was detected by the presence of circulating anti-hGAAantibodies. Antibody titers were highest at eleven weeks post-injectionwith an average of 16.08±4.66-fold above background levels. Afterfifteen weeks, antibody titers dropped significantly to 4.72±1.28-foldabove background and were further reduced to background levels by 31weeks post-treatment. Peak GAA enzyme activity levels were detected at24 weeks with 4223±1323% and 138.18±59.7% of normal (Gaa^(+/+)) activityin heart and diaphragm, respectively, with levels dropping to593.79±197.35% and 39.81±17.43% of normal, respectively, at one yearpost-injection.

Recombinant AAV2/1-Mediated Therapy Can Correct Cardiac Mass andConductance Abnormalities in Gaa^(−/−) Mice

The results described above demonstrate that delivery ofrAAV2/1-CMV-hGAA vectors results in sustained biochemical andhistological correction of the Pompe disease cardiac phenotype asevidenced by supraphysiologic levels of GAA enzyme activity and theconcomitant clearance of glycogen, as determined by periodicacid-Schiff's reagent staining. Proton magnetic resonance spectroscopy(¹H-MRS) of perchloric acid extracts of cardiac tissues furthersupported these findings. As shown in FIG. 11, a pronounced glycogenpeak could be detected in a 1-year-old Gaa^(−/−) mouse. An average 70%reduction was observed in the glycogen content in hearts of 1-year oldGaa^(−/−) mice treated with rAAV2/1-CMV-hGAA as neonates, as compared tountreated mice.

The physiological effects of rAAV2/1-mediated therapy on cardiacfunction were examined. A shortened P-R interval is characteristic inelectrocardiograms of patients with Pompe disease. At one year of age,Gaa^(−/−) mice also display a significantly shortened P-R interval. Asshown in Table 1, one-year-old Gaa^(−/−) mice that were administeredrAAV2/1-CMV-hGAA as neonates demonstrated significantly improved cardiacconductance with a prolonged P-R interval of 39.32±1.6 ms, as comparedto untreated controls (35.58±0.57 ms) (p<0.05). In addition to aberrantcardiac conductance, both the patient population and mouse model ofPompe disease also exhibit pronounced cardiac hypertrophy. Usingmagnetic resonance imaging (MRI), previous studies have shown thatcardiac mass can be accurately quantified, noninvasively, in mousemodels. MRI was used to assess left ventricular (LV) mass in theGaa^(−/−) model. At one year of age, Gaa^(−/−) mice have significantlyhigher LV mass (181.99±10.7 mg) as compared to age-matched wild-typeGaa^(+/+) (C57BL6/129SvJ) mice (140.79±5.12 mg). As shown in Table 1,rAAV2/1-treated Gaa^(−/−) mice have LV masses similar to that ofwild-type mice at one year of age (141.97±19.15 mg). While the reducedLV mass in the rAAV2/1-treated mice was not quite statisticallysignificant (p=0.06), the trend of smaller LV mass is thought to be realand would likely be significant with a larger sample population.

TABLE 1 Intravenous Injection of rAAV2/1 Leads to Decreased Cardiac Massand Elongated P-R Interval Ventricular Mass (mg) P-R Interval (ms) 1year-old Gaa^(−/−) 181.99 ± 10.70 35.58 ± 0.57 1 year-old BL6/129 140.79± 5.12  45.13 ± 1.16 1 year-old AAV2/1-treated  141.97 ± 19.15**  39.34± 1.60* One year post-injection, rAAV2/1-treated mice (n = 7) as well asuntreated age-matched control Gaa^(−/−) (n = 7) and C57 (n = 5) micewere subjected to electrocardiography as well as magnetic resonanceimaging.Diaphragm Contractility and Ventilatory Function are SignificantlyImproved After Administration of rAAV2/1 Vectors

As respiratory insufficiency manifests as one of the most prevalentclinical complications of Pompe disease, the effects of rAAV2/1-mediatedgene therapy on ventilatory function was examined in Gaa^(−/−) mice(Kishnani, P. S. et al., Genet. Med., 8:267-288, 2006; Hagemans, M. L.et al., Neurology, 66:581-583, 2006; Mellies, U. et al., Neurology,64:1465-1467, 2005). PAS staining of diaphragm from one-year-oldGaa^(−/−) mice administered rAAV2/1-CMV-hGAA intravenously, showed asignificant reduction in the amount of accumulated glycogen,corresponding with the therapeutic level of GAA expression. Diaphragmmuscle was isolated and assessed for isometric force generation.Diaphragm contractile force generated by rAAV2/1-treated mice wassignificantly improved as compared to age-matched, untreated controls,and even younger, 3-month-old untreated animals. At the maximalstimulation frequency (200 Hz), the force generated by diaphragms fromrAAV2/1-treated mice was 21.98±0.77 N/cm², whereas control one-year-oldGaa^(−/−) mouse diaphragms generated an average of 13.95±1.15 N/cm².

To measure ventilation, barometric whole-body plethysmography was used.Plethysmography allows for the simultaneous measurement of multipleparameters of ventilation, including frequency (breaths/min), tidalvolume (mL/breath), minute ventilation (mL/min), and peak inspiratoryflow (mL/sec), in unanesthetized, unrestrained mice (DeLorme, M. P. andMoss, O. R., J. Pharmacol. Toxicol. Methods, 47:1-10, 2002). Mice weresubjected to 90 min of normoxic air followed by a ten minute exposure tohypercapnic (7% CO₂) conditions. The elevated CO₂ levels increases thedrive to breathe and allows for an assessment of an extended range ofrespiratory capabilities. Plethysmography was performed at 6 and 12months of age. Untreated Gaa^(−/−) mice showed dramatically diminishedventilatory capacity at both 6 and 12 months of age, as demonstrated bysignificantly reduced frequency, tidal volume, minute ventilation, andpeak inspiratory flow (p<0.01) in response to hypercapnia. Conversely,at 6 months, rAAV2/1-treated Gaa^(−/−) mice had significantly improvedventilation across all parameters measured in response to hypercapnia(FIG. 12A-FIG. 12D), and at one year post-treatment, frequency, minuteventilation, and peak inspiratory flows were still significantly higherthan that of untreated age-matched controls (p<0.05) (FIG. 13A-FIG.13D).

The experiments described herein demonstrate that in addition tobiochemical correction of the disease phenotype, administration of atherapeutic rAAV2/1 vector can lead to functional correction as well.Treatment with a therapeutic rAAV2/1 vector resulted in a significantimprovement in cardiac function as indicated by an elongated P-Rinterval in electrocardiograms of treated animals.

These experiments also demonstrate that an average of approximately 39%normal GAA activity can result in clearance of glycogen in thediaphragm, the major muscle involved in ventilation, as well as adramatic improvement in the contractile capability of the diaphragm.Furthermore, a significant improvement of ventilatory function wasobserved under conditions of hypercapnia. Similar to the cardiacfunction, while marked improvement is noted in ventilatory function, thecorrection is only partial. A significant difference in ventilationbetween the treated animals and respective untreated controls duringexposure to normoxic conditions was not observed.

Experiments performed in the Gaa^(−/−) mouse model suggest that phrenicmotoneuron activity in Gaa^(−/−) mice is attenuated and demonstrate thata single intravenous administration of a therapeutic rAAV2/1 vector cangive rise to sustained correction of the cardio-respiratory phenotype ina mouse model of metabolic muscular dystrophy.

EXAMPLE 6 Gel-Mediated Delivery of rAAV2/1 Vectors to CorrectVentilatory Function in Pompe Mice with Progressive Forms Disease

The consequences of a gel-mediated method of delivery of a therapeuticrecombinant AAV2 viral vectors pseudotyped with viral capsids ofserotype 1 (rAAV2/1) in Gaa^(−/−) mice treated at 3, 9, and 21-months ofage was characterized. In mice treated at 3 months of age, a significantimprovement in diaphragm contractile strength at 6 months that issustained out to 1 year of age was observed compared to age-matcheduntreated controls. Similarly, significantly improved contractilestrength was observed in mice treated at 9 and 21 months of age, 3months post-treatment (p≦0.05). Ventilation under normoxic conditions(the ratio of tidal volume/inspiratory time, the ratio of minuteventilation to expired CO₂, and peak inspiratory flow) were all improvedin mice treated at 3 months of age and tested at 6 months (p≦0.05), butwas not sustained at 1 year of age, as compared to untreated age-matchedcontrols. In all rAAV2/1 gel-treated mice (treated 3, 9, and 21 monthsof age) minute ventilation and peak inspiratory flows were significantlyimproved under hypercapnic conditions. These results demonstrate that ingel-mediated delivery of rAAV2/1 vectors can mediate significantphysiological improvement of ventilatory function in a model of musculardystrophy.

Materials and Methods Packaging and Purification of Recombinant AAV2/1Vectors

The recombinant AAV2 plasmid p43.2-GAA has been described previously.Recombinant AAV particles based on serotype 1 were produced usingp43.2-GAA and were generated, purified, and titered at the University ofFlorida Powell Gene Therapy Center Vector Core Lab.

In vivo Delivery

All animal studies were performed in accordance with the guidelines ofthe University of Florida Institutional Animal Care and Use Committee.Three, nine, and 21-month-old Gaa^(−/−) mice were administered 1×10¹¹particles rAAV2/1-CMV-GAA directly to the diaphragm in a gel matrix asdescribed previously (see U.S. patent application Ser. No. 11/055,497filed Feb. 10, 2005).

Histological Assessment of Glycogen Clearance

Segments of treated and untreated diaphragm were fixed overnight in 2%glutaraldehyde in PBS, embedded in Epon 812® (Shell), sectioned, andstained with PAS by standard methods.

Assessment of Diaphragm Contractile Force

Isometric force-frequency relationships were used to assess diaphragmcontractile force. The diaphragm is isolated, with the ribs and centraltendon attached, and placed in Krebs-Henseleit solution equilibratedwith a 95% O₂/5% CO₂ gas mixture on ice. A single muscle strip, cut fromthe ventral costal diaphragm parallel to the connective tissue fibers,is used to determine force-frequency relationships. Plexiglas® clampsare attached to the diaphragm strip via clamping to the rib and centraltendon. The muscle strip is suspended vertically in a water-jacketedtissue bath (Radnoti, Monrovia, Calif.) containing Krebs-Henseleitsolution equilibrated with a 95% O₂/5% CO₂ gas mixture, maintained at37° C., pH 7.4, and equilibrated for 15 min. To measure isometriccontractile properties, the clamp attached to the central tendon isconnected to a force transducer (Model FT03, Grass Instruments, WestWarwick, R.I.). The transducer outputs are amplified and differentiatedby operational amplifiers and undergo A/D conversion using acomputer-based data acquisition system (Polyview, Grass Instruments). Todetermine the muscle strip optimal length (L_(o)) for isometric tetanictension, the muscle is field-stimulated (Model S48, Grass Instruments)along its entire length using platinum wire electrodes. Single twitchcontractions are evoked, followed by step-wise increases in musclelength, until maximal isometric twitch tension is obtained. Allcontractile properties are measured isometrically at L_(o). Peakisometric tetanic force is measured at 10, 20, 40, 80, 100, 150, and 200Hz. Single 500-ms trains are used, with a four-minute recovery periodbetween trains to prevent fatigue. Calipers are used to measure L_(o)before removal of the muscle from the apparatus. The muscle tissue isthen dissected away from the rib and central tendon, blotted dry, andweighed. The muscle cross-sectional area (CSA) is determined using theequation:

CSA (cm²)=[muscle strip mass (g)/fiber length L_(o) (cm)×1.056 (g/cm³)],

where 1.056 g/cm³ is the assumed density of muscle. The calculated CSAis used to normalize isometric tension, which is expressed as N/cm².

Assessment of Ventilatory Function

Ventilatory function was assayed using barometric whole bodyplethysmography. Unanesthetized, unrestrained C57BL6/129SvJ (n=10),Gaa^(−/−) (n=10), and rAAV2/1-treated Gaa^(−/−) mice (n=8) are placed ina clear Plexiglas® chamber (Buxco, Inc., Wilmington, N.C.). Chamberairflow, pressure, temperature, and humidity are continuously monitoredand parameters such as frequency, minute ventilation, tidal volume, andpeak inspiratory flow are measured and analyzed using the method byDrorbaugh and Fenn and recorded using BioSystem XA software (Buxco,Inc.). Baseline measurements are taken under conditions of normoxia(F₁O₂: 0.21, F₁CO₂: 0.00) for a period of one hour followed by a tenminute exposure to hypercapnia (F₁O₂: 0.93, F₁CO₂: 0.07).

Efferent Phrenic Nerve Recordings

Mice were anesthetized with 2-3% isoflurane, trachea canulated, andconnected to a ventilator (Model SAR-830/AP, CWE, Incorporated).Ventilator settings were manipulated to produce partial pressures ofarterial CO, between 45-55 mmHg. A jugular catheter (0.033 outerdiameter; RenaPulse™ tubing, Braintree Scientific) was implanted andused to transition the mice from isoflurane to urethane (1.0-1.6 g/kg)anesthesia. A carotid arterial catheter (mouse carotid catheter,Braintree Scientific) was inserted to enable blood pressure measurements(Ohmeda P10-EZ) and withdrawal of 0.15-mL samples for measuring arterialPO, and PCO₂ (I-Stat portable blood gas analyzer). Mice were vagotomizedbilaterally and paralyzed (pancuronium bromide; 2.5 mg/kg, i.v.). Theright phrenic nerve was isolated and placed on a bipolar tungsten wireelectrode. Nerve electrical activities were amplified (2000×) andfiltered (100-10,000 Hz; Model BMA 400, CWE, Incorporated). Whenmonitoring spontaneous inspiratory activity in the phrenic neurogram,the amplified signal was full-wave rectified and smoothed with a timeconstant of 100 ms, digitized and recorded on a computer using Spike2software (Cambridge Electronic Design; Cambridge, UK). The amplifiergain settings and signal processing methods were identical in allexperimental animals. The 30 seconds prior to each blood draw wereanalyzed for the mean phrenic inspiratory burst amplitude from thesedigitized records.

Results:

Gel-Mediated Delivery of rAAV2/1 Can Result in Efficient Transduction ofDiaphragm and Clearance of Accumulated Glycogen

Histological analysis of transduced diaphragms from mice administered1×10¹¹ particles rAAV encoding CMV promoter-driven β-galactosidase(lacZ) showed that not only could administration of rAAV2/1 lead touniform transduction across the surface of the diaphragm on which thevector was applied, but that rAAV2/1 vector could transduce the entirethickness of the diaphragm tissue. In comparison, rAAV2 vectors coldonly and transduce the first few layers of cells.

1×10¹¹ particles of rAAV2/1-CMV-GAA were administered to diaphragms ofadult three, nine, and 21-month-old Gaa^(−/−) mice using the gel method.GAA enzyme activity was assessed three months post-treatment for eachage group and in an additional cohort of mice treated at three months ofage, diaphragmatic GAA activity was assessed at nine monthspost-treatment. An average of 84.97±38.53% normal GAA activity wasobserved in treated diaphragms. No significant difference in GAAactivity was seen with respect to age at treatment, or with timepost-treatment as in the case of mice treated at three months of age andanalyzed at 6 month and 1 year of age, respectively. Periodicacid-Schiff (PAS) staining of diaphragm tissue also revealed a reductionin the amount stored glycogen in the tissue for all treated age groups.

Diaphragm Contractility is Significantly Improved After Administrationof rAAV2/1 Vectors

Similar to the Pompe patient population, Gaa^(−/−) mice have aprogressive weakening of diaphragm contractile strength correlating withduration of disease. Isometric force-frequency relationships fromdiaphragm muscle isolated from untreated Gaa^(−/−) mice (n=3 for eachgroup) show a significant decrease in contractile strength with age from3 months of age to 2 years of age. After gel-mediated administration ofrAAV2/1-CMV-hGAA to diaphragms of Gaa^(−/−) mice, a significantimprovement was seen in the contractile strength in the diaphragm muscleas compared to age-matched untreated controls. For animals that weretreated at 3 months of age, significantly improved diaphragm contractilestrength at 6 months (peak force of 24.83±3.31 N/cm²) was sustained outto 1 year (21.59±1.59 N/cm²) of age, as compared to age-matcheduntreated controls (peak force of 16.53±0.74 and 13.94±1.15 N/cm² at 6months and 1 year, respectively). In mice treated at 9 months (peakforce of 21.28±1.49) and 21 months (peak force of 17.21±0.29) of age, asignificant improvement was still seen in contractile function oftreated diaphragms, 3 months post-treatment as compared to age-matcheduntreated controls (peak force of 12.71±0.94 at 2 years of age).

Ventilatory, Function is Improved After Administration of rAAV2/1Vectors to Adult Pompe Mice

Using barometric plethysmography, multiple characteristics ofventilation in conscious, unrestrained mice were simultaneouslymeasured. In this study, ventilation was measured under conditions ofnormoxia (normal breathing air oxygen levels; F₁O₂: 0.21, F₁CO₂: 0.00)and to assess the extended range of ventilatory capacity, underconditions of hypercapnia (higher than normal levels of carbon dioxide;F₁O₂: 0.93, F₁CO₂: 0.07).

Under conditions of normoxia, ventilation (the ratio of tidalvolume/inspiratory time (V_(T)/Ti; mL/sec) (2.2±0.1 vs 1.79±0.16), theratio of minute ventilation (mL/min) to expired CO₂ (V_(E)/VCO₂)(18.65±0.73 vs 13.3±0.74), and peak inspiratory flow (mL/sec) (4.11±0.17vs 3.21±0.29)) were all improved (p≦0.05) in mice treated at 3 months ofage and tested at 6 months as compared to untreated age-matchedcontrols. Correction of ventilatory function in normoxic conditions wasnot sustained though, as none of the parameters were significantlyimproved at one year of age (9 months post-treatment). Animals that weretreated at 9 months and 21 months of age also did not show improvednormoxic ventilation three months post-treatment. Conversely,hypercapnic respiratory challenge resulted in improved ventilatoryfunction in all treated groups. As shown in FIG. 14A-FIG. 14D and FIG.15A-FIG. 15D, for animals treated at 3 months and assayed at 6 monthsand 1 year of age as well as in animals treated at nine months and 21months of age, minute ventilation (FIG. 14A-FIG. 14D) and peakinspiratory flow (FIG. 15A-FIG. 15D) were significantly increased overage-matched untreated control animals.

Increased Phrenic Nerve Activity After Gel-Mediated Delivery of rAAV2/1to Gaa Mouse Diaphragm

It was of interest to examine the phrenic nerve activity in an animaladministered rAAV2/1 to the diaphragm muscle. As shown in FIG. 16, theinspiratory phrenic burst amplitude in a 2-year-old Gaa^(−/−) mouseadministered rAAV2/1-CMV-hGAA via the gel method at 21 months of age wasgreater than that of an age-matched, untreated control animal,suggesting a possible correction of the potential neural deficits inPompe disease.

Due to the physical nature of the mouse diaphragm (size and thickness),a gel-based method of vector delivery was used. rAAV2/1 vector couldspread through the thickness of the diaphragm, whereas rAAV2 vectorcould only transduce the first few cell layers. The spread of vector maybe attributed to the capsid conferring differential infection viacellular receptors and/or trafficking through the tissue via the processof transcytosis.

In this study, direct administration of rAAV2/1 vector to the diaphragmresulted in increased phrenic nerve activity in the treated animal ascompared to an untreated control. Taken together these results indicatethat physiological correction of diaphragm function can be mediated byrAAV2/1-based gene therapy and that even older animals as old as 21months of age (note that the average lifespan of a wild-type C57BL mouseis approximately 2 years of age) can benefit from gene therapytreatment.

EXAMPLE 7 Neural Deficits Contribute to Respiratory Insufficiency in aMouse Model of Pompe Disease

Respiratory dysfunction is a hallmark feature of Pompe disease andmuscle weakness is viewed as the underlying cause, although thepossibility of an associated neural contribution has not heretofore beenexplored. In the experiments described herein, behavioral andneurophysiological aspects of breathing in an animal model of Pompedisease—the Gaa^(−/−) mouse—and in a second transgenic line (MTP)expressing GAA only in skeletal muscle were examined. Glycogen contentwas significantly elevated in Gaa^(−/−) mouse cervical spinal cord,including in retrogradely labeled phrenic motoneurons. Ventilation,assessed via barometric plethysmography, was attenuated during bothquiet breathing and hypercapnic challenge in Gaa^(−/−) mice (6 to >21months of age) vs. wild-type controls. MTP mice had normal diaphragmaticcontractile properties; however, MTP mice had ventilation similar to theGaa^(−/−) mice during quiet breathing. Neurophysiological recordingsindicated that efferent phrenic nerve inspiratory burst amplitudes weresubstantially lower in Gaa^(−/−) and MTP mice vs. controls. It wasconcluded that neural output to the diaphragm is deficient in Gaa^(−/−)mice, and therapies targeting muscle alone may be ineffective in Pompedisease.

Methods Animals

The Gaa^(−/−) and muscle-specific hGAA (MTP) mice have been previouslydescribed (Raben et al., Hum. Mol. Genet., 10:2039-2047, 2001; Raben etal., J. Biol. Chem., 273:19086-19092, 1998). Contemporaneous gendermatched C57B1/6 X 129X1/SvJ mice were used as controls for allexperiments. Mice were housed at the University of Florida specificpathogen-free animal facility. The University of Florida's InstitutionalAnimal Care and Use Committee approved all animal procedures.

Barometric Plethysmography

Barometric plethysmography to quantify ventilation (Buxco Inc.,Wilmington, N.C.) has been described previously and was adapted formice. Ventilation was characterized in male and female mice. Genderswere separated only when significant differences were detected betweenmale and female mice. Data from a subset of the animals used in theseexperiments have been reported as controls for a gene therapyintervention.

Hemoglobin, Hematocrit, Glucose and Sodium Blood Levels

Venous tail blood was collected from anesthetized mice (2% isoflurane,balance O₂) directly into a commercially available blood gas analysescartridge (I-stat, Heska Corporation; Ft. Collins, Colo.).

Retrograde Labeling of Phrenic Motoneurons

The neuronal retrograde tracer Fluoro-Gold® (4%, Fluorochrome, LLC,Denver, Colo.) was applied to the peritoneal surface of the diaphragm(˜75 μL) using a small artist's brush. Care was taken to apply thetracer sparingly only to the diaphragm in order to minimize leakage toliver and surrounding tissues. Forty-eight hours after Fluoro-Gold®application, the cervical spinal cord (C₃-C₅) was removed,paraffin-embedded and sectioned in the transverse plane at 10 μm.Fluoro-Gold®-labeled phrenic motoneurons were identified by fluorescencemicroscopy.

Statistics

Statistical significance for this project was determined a priori atp<0.01. Ventilation data were analyzed using a 3-way analysis ofcovariance (ANCOVA). Ratios of volume:bodyweight were not used, as bodymass ratios can introduce bias and this method does not have theintended effect of removing the influence of body mass on the data. Byusing the ANCOVA method, bodyweight is analyzed as a co-variate for allrespiratory volume data, which more accurately removes the influence ofbodyweight on the data. For baseline measures, gender, strain and agewere used as factors while the hypercapnic data was analyzed usinggender, strain and time (minutes 1-10 of hypercapnia) as factors.Hemoglobin, hematocrit, glucose and sodium (anesthetized mice) wereanalyzed using the student's t-test. Glycogen quantification wasanalyzed using a 2-way ANOVA and t-test with Bonferroni correction forpost-hoc measurements. Diaphragmatic muscle contractile function wasanalyzed using a 2-way ANOVA with repeated measures. Phrenic inspiratoryburst amplitude, breathing frequency and the rate of rise of the phrenicburst were extracted from the phrenic neurogram. These variables andarterial P_(a)CO₂ were analyzed with the 1-way ANOVA and Fischer's LSDtest for post-hoc analysis. All data are presented as the MEAN±SEM.

Arterial blood sampling, glycogen quantification, histological glycogendetection in motoneurons, in vitro diaphragmatic contractile properties,and efferent phrenic nerve recordings were performed as described(Martineau, L, and Ducharme, M. B., Contemp. Top. Lab. Anim. Sci., 37(5):67-72, 1998; Lo et al., J. Appl. Physiol., 28:234-236, 1970; Guth,L, and Watson, P. K., Exp. Neurol., 22:590-602, 1968; Staib et al., Am.J. Physiol. Regul. Integr. Comp. Physiol., 282 (3):R583-90, 2002;Doperalski, N. J. and Fuller, D. D., Exp. Neurol., 200 (1):74-81, 2006).

Results General Features of Gaa^(−/−) Mice

Gaa^(−/−) mice weighed significantly less than their wild-type controlsat all ages. No age-related gender differences were observed, and malesweighed significantly more than females at all ages.

Glycogen Quantification and PAS Staining of the Cervical Spinal Cord

Glycogen content was elevated at all ages in the cervical spinal cords(C₃-C₅) of Gaa^(−/−) mice, and differences were more pronounced at >21vs. 6 months (FIG. 17A). These data were confirmed in an independentseries of experiments in which glycogen levels were determined inmultiple levels of the neuraxis.

Correlative histochemistry also demonstrated significant glycogenreaction product in Gaa mouse neuronal cell bodies throughout the graymatter of the cervical spinal cord that was especially prominent inmotoneurons (FIG. 17E, FIG. 17F and FIG. 17G). Motoneurons in theventral cervical spinal cord retrogradely labeled with Fluoro-Gold®exhibited prominent PAS droplets (positive glycogen) throughout the cellbody cytoplasm (FIG. 17G). Comparable neurons from PAS-stained sectionsof control specimens showed neurons with virtually no PAS-positiveinclusions (FIG. 17B, FIG. 17C and FIG. 17D).

Ventilation

Gaa^(−/−) mice appeared to be hypoventilating based on the minuteventilation/expired CO₂ ratio, which normalizes minute ventilation tometabolic CO₂ production. This measure was attenuated at baseline inGaa^(−/−) mice vs. wild-type controls. Baseline minute ventilation(non-normalized), breathing frequency, tidal volume, peak inspiratoryflow, peak expiratory flow and tidal volume/inspiratory time ratio werealso decreased in Gaa^(−/−) mice compared to controls at all agesstudied (Table 2, FIG. 18). The only age differences detected were lowerfrequency at >21 months (vs. 6 months) and elevated tidal volume at >21months (vs. 6 months). No strain by age interaction was detected in theanalyses.

TABLE 2 Baseline Ventilation Characteristics Frequency MV PIF PEF TV/T(breaths/min) TV (mL/breath) (mL/breath) (mL/sec) (mL/sec) (mL/sec) 6month Control: 239 +/− 7 0.27 +/− 0.00 64.8 +/− 3.7 5.9 +/− 0.2 3.4 +/−0.2 3.4 +/− 0.2 Gaa^(−/−): 197 +/− 6* 0.21 +/ 0.00*  41.6 +/− 2.3*  3.3+/− 0.2*  2.2 +/− 0.1*  1.7 +/− 0.1* 12 month Control: 252 +/− 7 0.31+/− 0.00 77.3 +/− 3.4 6.7 +/− 0.2 4.4 +/− 0.2 3.9 +/− 0.2 Gaa^(−/−): 186+/− 7* 0.23 +/− 0.00*  43.2 +/− 2.3*  3.6 +/− 0.1*  2.3 +/− 0.1*  2.1+/− 0.1* >21 month Control: 225 +/− 7 

0.33 +/− 0.00 

73.4 +/− 3.2 6.3 +/− 0.3 4.6 +/− 0.2 3.7 +/− 0.2* Gaa^(−/−): 168 +/− 7* 

0.25 +/− 0.01* 

 41.8 +/− 3.8*  3.5 +/− 0.3*  2.3 +/− 0.2* 2.1 +/− 0.2* Sixty minutebaseline (21% O₂, balanced N₂) values for frequency, tidal volume (TV),minute ventilation (MV), peak inspiratory flow (PIF), peak expiratoryflow (PEF) and tidal volume: inspiratory time ratio (TV/Ti) of controland Gaa−/− mice. *= Gaa−/− different from control (p < 0.01).

 = >21-month different from 6 months (p < 0.01).

Hypercapnic challenge was used as a respiratory stimulus to test thecapacity to increase ventilation in Gaa^(−/−) mice. The hypercapnicresponse was lower for Gaa^(−/−) mice vs. controls at each age forminute ventilation (FIG. 18A and FIG. 18B), as well as frequency, tidalvolume, peak inspiratory flow, peak expiratory flow and the tidalvolume/inspiratory time ratio. Gender differences were detected only inthe 6 month age group, whereby females had a different response tohypercapnia for all respiratory variables tested.

Blood Sampling

Both Hemoglobin (Hb) and Hematocrit (Hct) were elevated in Gaa^(−/−)mice (Table 3), most likely to compensate for insufficient arterialpartial pressure of O₂ (P_(a)O₂; see below). In addition, glucose andsodium levels did not vary between control and Gaa^(−/−) mice,suggesting that the measured Hb and Hct differences did not reflectplasma volume differences. Gaa^(−/−) mice had lower P_(a)O₂ vs. controls(Table 3), supporting the concept that these mice are hypoventilating.

TABLE 3 Blood Characteristics for 12 Month Gaa^(−/−) and Control MiceHEMOGLOBIN HEMATOCRIT SODIUM GLUCOSE P_(a)O₂ (g/dL) (%) (mmol/L) (mg/dL)(mmHg) CONTROL: 13.5 ± 0.3  39.8 ± 0.9  144.5 ± 0.8 180.4 ± 16.3 98.5 ±1.9  Gaa^(−/−) 15.3 ± 0.4* 45.0 ± 1.1* 143.4 ± 0.9 176.8 ± 11.4 83.3 ±2.7* 2.7* Hemoglobin, hematocrit, sodium and glucose values for controland Gaa^(−/−) mice at 12 months of age (n = 9/group). Arterial partialpressure of O₂ for control and Gaa^(−/−) mice at 12 months of age (n =6/group). *= Gaa^(−/−) different from control (p <0.01).Muscle-Specific hGAA Mice

Next, respiratory function in transgenic animals with muscle-specificcorrection of GAA activity (MTP mice) was quantified. To first obtain anindex of diaphragm muscle function, in vitro contractile properties fromB6/129, Gaa^(−/−) and MTP mice were measured. Control and MTP mice hadsimilar forces, while the Gaa^(−/−) mice produced significantly smallerforces. These data confirm that the normal glycogen levels in MTPdiaphragm muscle (MTP vs. B6/129; 1.71±1.3 vs. 1.4±0.2 μg/mgww)correspond to diaphragm muscle that is functionally similar to theB6/129 mice.

Despite apparently normal functional diaphragm muscle (FIG. 19C), thepattern of breathing was altered in the MTP mice. Minute ventilationduring baseline was similar in MTP and Gaa^(−/−) mice, and both weresignificantly reduced compared to B6/129 mice (FIG. 19A-FIG. 19C).Furthermore, the response to hypercapnia was attenuated in MTP mice,although they showed a greater response than the Gaa^(−/−) mice (FIG.19B). Representative airflow tracings from B6/129, Gaa^(−/−) and MTPmice (FIG. 20C) were generated (FIG. 19C).

Efferent Phrenic Activity

To determine whether the compromised ventilation seen in Gaa^(−/−) andMTP was associated with reduced phrenic motor output, we measuredefferent phrenic nerve activity in Gaa^(−/−), MTP and control mice. Atsimilar arterial PCO₂ levels (see legend, FIG. 20A), Gaa^(−/−) and MTPmice had significantly lower phrenic inspiratory burst amplitudes (FIG.20A and FIG. 20B). The neurogram recordings from Gaa^(−/−) and MTP micealso revealed less frequent bursts, and an attenuated slope of theintegrated inspiratory burst (i.e. slower “rate of rise”, Table 4).

TABLE 4 Phrenic Neurophysiology Characteristics Rate 

of 

Rise 

(mV/

Control: 346 ± 86  Gaa^(−/−):  44 ± 15* MTP: 101 ± 27* Rate of rise forthe phrenic burst (mV/s), frequency of the phrenic burst (neuralbreaths/s) and amplitude of the phrenic burst (mV) for 12 month oldcontrol (n = 8), Gaa^(−/−) (n = 8) and MTP (n = 6) mice. *= differentfrom control (p < 0.01)

indicates data missing or illegible when filed

This study of a murine model of Pompe disease has revealed severalobservations pertaining to GAA-deficiency and concomitant respiratoryinvolvement. First, ventilation is reduced in Gaa mice as revealed bybarometric plethysmography. Second, cervical spinal cord glycogen iselevated in Gaa^(−/−) mice, and PAS staining identified prominentglycogen inclusions in cervical motoneurons, including phrenicmotoneurons indirectly identified by retrograde Fluoro-Gold® tracing.Third, Gaa^(−/−) mice have attenuated phrenic output relative towild-type controls. Lastly, MTP mice also exhibit breathing impairmentsand phrenic neurogram features similar to those observed in Gaa^(−/−)mice, despite apparently normal diaphragmatic contractile function (FIG.21A and FIG. 21B). These are the first formal lines of evidencesuggesting respiratory weakening in the Gaa^(−/−) mouse, and byextrapolation in Pompe disease patients, may be the result of acombination of both neural and muscular deficits.

Excess glycogen within the spinal cord (including phrenic motoneurons)led to the quantification of inspiratory phrenic burst amplitude betweencontrol and Gaa−/− mice in the experiments described herein. Phrenicnerve activity, which is the final motor output of the respiratorysystem, was measured. The mechanisms responsible for the reduced outputin Gaa^(−/−) mice could stem from areas beyond the phrenic motoneurons,which include higher (neural) respiratory inputs and/or impairment ofchemosensory afferents due to chronically attenuated PaO₂ levels and thehypothesized elevated PaCO₂ levels. However, it should be noted thatduring conditions of higher respiratory drive (PaCO₂˜90 mmHg) bothgroups were able to increase phrenic inspiratory burst amplitude, butthe Gaa^(−/−) mice continued to have lower output (control: 68.7mv±20.0, Gaa^(−/−): 14.0 mV±4.8). The final end product of the phrenicnerve activity was altered in Gaa^(−/−) mice thus demonstrating a neuraldeficit of respiratory control.

To determine that muscular dysfunction was not the only contributor toventilation deficits due to GAA deficiency, a double transgenic mousethat expressed hGAA only in skeletal muscle (maintained on the Gaa^(−/−)background) was used. Since these mice had normal muscle contractileproperties, it was hypothesized that any differences in ventilationbetween MTP and control strains would reflect disparity in the neuralcontrol of respiratory muscles. Consistent with this postulate,ventilation was similar between MTP and Gaa^(−/−) mice during quietbreathing. When respiratory drive was stimulated with hypercapnia, theventilatory response of MTP mice was still less than that of controls,but elevated compared to Gaa^(−/−) mice. Thus, both muscle and neuralcomponents contribute to ventilation deficits under conditions ofelevated respiratory drive.

EXAMPLE 8 AAV Administered to Muscle is Able to be Transported to theMotor Nerve Body Via the Synapse with the Muscle Fiber

Referring to FIG. 22, FIG. 23 and FIG. 24, AAV administered to muscle isable to be transported to the motor nerve body via the synapse with themuscle fiber. In experiments in which mice received intrathoracicinjection of AAV-CMV-LacZ (2.18×10¹¹ particles) (FIG. 22), genomic DNAisolated from diaphragm contains control gene post vector delivery. Inexperiments in which mice received intrathoracic injection ofAAV-CMV-LacZ (2.18×10¹¹ particles), (FIG. 23), genomic DNA was isolatedfrom the phrenic nucleus. FIG. 24 shows that ventilation is improved 4weeks post-injection with AAV-CMV-GAA (2.52×10¹⁰ particles).

EXAMPLE 9 Restoration of the Neuromuscular Junction Integrity withrAAV2/9 Vectors

In developing therapies for muscular dystrophies, there exists theunique challenge of achieving simultaneous widespread correction of allaffected tissues.

This Example shows that the rAAV2/9 vector encoding hGAA results inretrograde transport of the transgene (human GAA) to motor neurons, aswell as effective transduction of skeletal muscles, CNS, and cardiactissues (FIG. 25). In addition, the administration of rAAV2/9-GAAvectors result in restoration of neuromuscular junction integrity, andthe reversal of axonal pathology in Pompe disease. The restoration ofadequate GAA levels in motor neurons provides significant improvement inventilatory parameters of Pompe animals.

(A) Direct Intramuscular or Intraspinal Injection

Briefly, one-year-old Pompe mice (Gaa^(−/−)) were randomized to thefollowing groups: untreated (Gaa^(−/−)), AAV2/9-CMV-hGAA, orAAV2/9-DES-hGAA. AAV2/9-treated animals received a single intramuscularinjection of 1×10¹¹ vg of vectors in the right tibialis anterior muscle.One month post injection, the tibialis anterior muscle and lumbar spinalcord were analyzed for vector genome copy number and GAA activity.

As shown in FIG. 26, high levels of vector genomes were detected in thetibialis anterior (AAV2/9-DES-hGAA 1.5×10⁵±3.1×10⁴ vg/ug DNA;AAV2/9-CMV-hGAA 8.4×10⁴±1.7×10⁴ vg/ug DNA) and lumbar spinal cord(AAV2/9-DES-hGAA 1.5×10³±1.7×10² vg/ug DNA; AAV2/9-CMV-hGAA2.5×10³±1.3×10³ vg/ug DNA), indicating efficient transduction ofskeletal muscle and retrograde transport of AAV2/9 vectors. Activity ofGAA in tibialis anterior lysates was 2396% and 1770% above wild-type inAAV2/9-DES-hGAA and AAV2/9-CMV-hGAA animals, respectively (p<0.05).Immunohistochemical assessment of neuromuscular junctions in thetibialis anterior revealed a restoration of integrity in AAV2/9-treatedanimals (FIG. 27).

In this Experiment, the tissue-specific expression of the transgene(GAA) is compared between the traditional CMV promoter and the moretissue-restricted Desmin (DES) promoter. Briefly, the human desminconstruct described in the experiments (AAV2/9-DES) contains both amyocyte specific enhancer factor 2 (MEF2) and a MyoD enhancer element.Analysis of human tissues revealed desmin expression in cerebellum,endometrium, skeletal muscle, neuronal cells of the lateral ventricleand heart. This is advantageous for AAV constructs as Desmin serves as anon-viral and tissue-restricted promoter compared to the traditional CMVpromoter (viral, ubiquitious cellular expression). In addition,expression profiles from the spinal cord also show that Desmin isexpressed, making it a favorable promoter for driving therapeutictransgenes by AAV vectors.

Enzymatic activity assays for GAA 28 days postinjection demonstratesignificant enzyme levels and vector genome copies in the TA and lumbarspinal cord (FIG. 25). Direct administration of AAV2/9 to skeletalmuscle results in efficient transduction of the injected muscle anddisplays retrograde transduction of lumbar motor neurons as shown byevidence of vector genome copies in the lumbar spinal cord region, aswell as restoration of neuromuscular junction (NMJ) integrity inGaa^(−/−) animals. The results also show that intramuscular delivery ofrAAV vectors transduces CNS and skeletal muscle tissues, and thus, canbe used to treat the CNS and skeletal muscle components of Pompedisease.

Materials and Methods

Packaging and purification of recombinant AAV2/9 vector.

Recombinant AAV particles based on serotype 9 were produced usingp43.2-GAA and were generated, purified, and titered at the University ofFlorida Powell Gene Therapy Center Vector Core Lab.

Experimental Animals.

The exon 6 GAA knockout animal model, which represents the cardiac andskeletal muscle phenotype associated with Pompe disease, was used toexamine the transduction efficiency of AAV2/9-mediated GAA (AAV2/9-GAA)therapy and vector construct specificity.

Gaa^(−/−)/129SvE (Gaa^(−/−)) mice were injected with either 20 μl oflactated ringer's solution or with 1×10¹¹ vg rAAV2/9-CMV-GAA orrAAV2/9-DES-GAA diluted in lactated ringer's solution (QS 20 μl).

Whole Mount Neuromuscular Junction.

Skeletal muscles were dissected and incubated in α-bungarotoxin (TRITClabeled) for 10 minutes. Washed and fixed in 4% paraformaldehyde.Tissues were blocked in 4% BSA (1% TritonX) and incubated with NFH.Secondary antibodies (Alexa 488) directed against NFH were performed andtissues were mounted for subsequent microscopy.

Sciatic Nerves.

Sciatic nerves were isolated and placed in cassettes for OCT fixation.Sections were cut at 5 microns and subjected to primary and secondary Abincubation with NFH, MBP, SMI-32 or H&E visualization.

Genomic DNA Extraction and Real-Time PCR.

PCR was used to measure distribution of AAV genomes followingintramuscular injection in the TA and lumbar spinal cord. DNA wasisolated using the DNAeasy kit (Qiagen) according to manufacturer'sinstructions. Primers and probes were designed to the SV40 poly-A regionof the AAV vector.

GAA Activity Assay.

Tissues were harvested, immediately snap frozen and stored at −80° C.Homogenates were centrifuged at 14,000 rpm for 10 minutes at 4° C. Theresulting supernatant was assayed for GAA activity by measuring thecleavage of 4-methylumbelliferyl-α-Dglucopyranoside after incubation for1 hour at 37° C. Protein concentration was measured using the Bio-Rad DCprotein assay kit per manufacturer's instructions. Data are expressedrelative to values measured in untreated GAA tissue levels (% WT).

(B) Direct Intraspinal Administration

To directly transduce the cervical cord region and phrenic motor neuronpool, rAAV-GAA vectors were administered into the C3-C5 region of thespinal cord of the Gaa^(−/−) mice via direct intraspinal injection.

As shown in FIG. 28, direct injection of rAAV2/9 reporter constructs inmouse C3-C5 region resulted in transduction of the phrenic motor neuronpool. Delivery of the therapeutic transgene (GAA) in Gaa^(−/−) animalssignificantly improved respiratory function, and the expression of GAAin the phrenic motor neuron pool was confirmed via immunohistochemicaldetection (FIG. 29). The observation of increased respiratory functionwas the result of CNS transduction alone, as vector genome copies werenot detected in the diaphragm. The results also show that glycogenaccumulation in the CNS impairs skeletal muscle function.

(C) Direct Intrathoracic Administration

With the observation that expression of GAA in the CNS vastly improvesrespiratory muscle function, experiments were conducted to target bothrespiratory muscle (e.g., diaphragm and costal) and the CNS (cervicaland thoracic regions) (FIG. 30). In these experiments, AAV2/9-CMV-GAAwas compared to AAV2/9-DES-GAA to examine the retrograde efficiency ofthe DES promoter in the CNS.

Briefly, Gaa^(−/−) animals received a single injection of either theAAV2/9-CMV-GAA or AAV2/9-DES-GAA vectors. The administration ofAAV2/9-DES-GAA resulted in similar or superior transduction efficiencyof target tissues when compared to AAV2/9-CMV-GAA, and resulted inimprovement in cardiac and respiratory function.

Intrathoracic injection in Gaa^(−/−) animals resulted in detection ofvector genomes in the diaphragm and in the C3-C5 region of the spinalcord (phrenic motor neuron region). The results show that a retrogradetransport phenomenon occurs with the use of AAV2/9 vectors.

To determine the physiological implications of transduction of bothrespiratory muscle and the CNS, Gaa^(−/−) animals received a singleintrathoracic injection of AAV2/9-CMV-GAA or AAV2/9-DES-GAA, and weresubjected to neurophysiological measurements 6 months post-injection.Direct in situ measurement of the phrenic nerve revealed an apparenttherapeutic effect by evidence of increased burst amplitude duringhypercapnic conditions and diaphragm EMG activity (FIG. 31).

In addition, experiments were conducted to determine whether retrogradetransport is evident in the phrenic motor neuron pool, which has asignificant impact on proper respiratory function. One of the hallmarksof Pompe disease is respiratory insufficiency. Although respiratoryinsufficiency is traditionally thought to be the result of respiratorymuscle weakness, the present inventors discovered that respiratoryinsufficiency could result from impairment in the activation of thephrenic nerve due to glycogen accumulation.

To directly visualize AAV-mediated retrograde transduction in thephrenic motor neuron pool, AAV2/9-GFP vectors were injected to Gaa^(−/−)animals via intrathoracic injections. One month following injection,animals were perfused and the entire spinal cord was fixed forcross-sectional immunohistochemical analysis for detection andlocalization of GFP expression.

As shown in FIG. 32, positive staining for GFP was detected in the C3-C5and T2-T5 regions of the spinal cord. The inset in each area isindicative of positive staining for the phrenic (left) and costal(right) motor pool, respectively. FIG. 33 shows that the administrationof rAAV-CMV-GAA and rAAV-DES-GAA improves phrenic nerve signalpropogation.

(D) Intravenous Administration

Intravenous administration of rAAV-GAA vectors provides treatment ofPompe disease, including correction of cardiac and respiratory muscledysfunction. Briefly, 4×10⁸ vg of rAAV2/9-CMV-hGAA vectors wereadministered to one-day-old Gaa^(−/−) mice via intravenously (IV)injection. Assessment of cardiac measurements (e.g., P-R interval,cardiac output, and ejection fraction) revealed a significantimprovement in treated Gaa^(−/−) mice as compared to untreated animalsout to 6 months post-injection. Additionally, as a result of restoredGAA activity, glycogen deposition was dramatically reduced in cardiacand diaphragm tissues of the treated animals.

In another experiment, three-month-old Gaa^(−/−) mice received a singleintravenous injection of 1×10¹¹ vg (˜5×10⁶ vg/kg) AAV2/9-(CMV orDES)-GAA. Cardiac and diaphragm functional measurements were conductedat 3 months post-treatment (6 months of age) in parallel to age-matcheduntreated Gaa−/− and wild-type 129SVe (WT) mice. DES-GAA treated animalsshowed a marked improvement in cardiac function with levels similar toWT (FIG. 34A) and reduction of left ventricular mass. Contractilefunction of diaphragm strips from untreated Gaa^(−/−), AAV2/9-CMV,AAV2/9-DES, and WT was measured, and the results show improved functionin AAV2/9/-CMV-GAA and AAV2/9-DES-GAA treated groups (FIG. 34F). Theresults also show that the DES-driven GAA is more efficacious insystemically treated animals.

EXAMPLE 10 Production of rAAV Vectors Using an HSV-Based System

One major hurdle for the use of rAAV in gene therapy applications is thelack of capability to generate high yield of vector at high titer andhigh purity. Manufacturing amounts in the range of 1×10¹⁴ to 1×10¹⁵vector genomes using the transfection-based production system remainscumbersome, time-consuming and costly.

This Example pertains to the production of large amount of rAAV vectors(e.g., rAAV2/9-hGAA) using a production platform based on the HerpesSimplex virus type I System (HSV). The current system utilizes tworecombinant HSV: one carrying the AAV rep and cap, or “helper” and theother one carrying the recombinant AAV genome containing the transgeneexpression cassette. AAV production is initiated upon co-infection ofcells with the two recombinant HSV and harvest typically occurs within52 hours. Producer cells (HEK293 or BHK21) can be grown on adherentsupport (flask or cell factory) or in suspension in a Wave Bioreactortype technology.

An additional challenge for the use of rAAV in gene therapy applicationsis the lack of efficient, high recovery streamline purification methodfor AAV vectors (e.g., AAV2/9). This Example also provides a streamlineprotocol to purify rAAV9 particles from crude cell extracts that can beapplied to large-scale production protocols.

Establishment of a scalable and flexible platform based on the HSVsystem is required for generating sufficient amounts of highly pure andconcentrated rAAV vectors (e.g., rAAV2/9-GAA). rAAV9 vector stocksprepared using the HSV system or the standard co-transfection protocolin 293 cells are compared based on the following parameters: i) thebiochemical purity of the preparation (i.e., absence of contaminantproteins) ii) the ratio of infectious-to-physical particles; iii) theratio of VP1:VP2:VP3 capsid proteins; and iv) the ratiofull-versus-empty particles.

Implementation of scalable production method based on the HSV system anda streamline purification protocol for AAV9 enables the preparation oflarge amounts of highly pure and potent rAAV vectors (e.g., rAAV2/9-GAA)in a time and cost effective manner. rAAV vectors (e.g., rAAV2/9-GAA)manufactured using the HSV system are characterized based on purity andbiological potency levels comparable to, or exceeding, the vectorsprepared in HEK293 cells using transfection method.

FIG. 35 is a schematic representation of vector constructs of theHSV-based production system. FIG. 36 shows streamlined purification ofrAAV vectors by pl precipitation and column chromatography. FIG. 37shows AAV production by HSV coinfection method using adherent cells.

Preparation of HSV-AAV-hGAA and HSV-AAV/rep2cap9 helper.

Two recombinant HSV-AAV-hGAAs (HSV-hGAA and HSV-coGAA) are engineered byhomologous recombination into the thymidine kinase (tk) locus of thereplication-defective rHSV-1 vector, ICP27-deleted (infected cellprotein 27). AAV-DES-hGAA contains the human GAA ORF downstream of thedesmin promoter and bracketed by AAV serotype 2 terminal repeats (ITRs).The AAV-DES-cohGAA contains a codon-optimized version of the human GAAORE for higher expression in eukaryotic cells (GeneArt, Invitrogen).

The HSV-AAV9 helper is generated by inserting the PCR generated sequenceof AAV2 rep, and AAV9 cap from plasmid pRep2Cap9 (obtained from Dr. JimWilson, UPenn Recombinant viruses is screened and isolated by plaqueassay and further amplified by clonal propagation on V27 cells,Vero-derived cells which supply ICP27.

Methods for generating rHSV.

HSV viruses are recovered from cell supernatant and are further filteredand concentrated prior to titering. Small and medium scale rAAVproductions are conducted in either HEK293 grown in flasks (T225 or cellfactory) or in suspension-adapted BHK cells in spinner cultures (up to 1L or ˜1×10⁹ cells). Cells are coinfected with rHSV-hGAA (or rHSV-cohGAA)and rHSV-rep2/cap9 WVBs at appropriate MOIs. Cells are harvested at ˜52hours post-infection. AAV9 are purified using our standard purificationmethod using freeze-thaws and iodixanol gradient, followed byconcentration using Apollo centrifugal device. Stocks are analyzed forvector genome titer by Q-PCR and for purity by silver stained SDS-PAGE.

An alternative purification method for mid-scale production (>1×10⁹cells) involves preparing cell crude lysates by microfluidization andpurifying AAV9 particles by pI precipitation and column chromatography.During Process & Development, methods described above as well as smallbatches of AAV9 prepared in HEK293 by conventional CaPO₄ transfectionare evaluated with respect to yield/cell, ratio empties/full, purity andratio particle-to-infectious units.

Scale-up of the suspension-based format is necessary for large scalerAAV9 production. Briefly, sBHK cells are grown in either spinnercultures (1 L and up) or disposable bioreactor type Wave 10 L and up(Wave™, GE Healthcare) and coinfected with rHSV-hGAA and rHSV-rep2/cap9WVBs. The advantages of the suspension BHK system is an increased celldensity (up to 2×10⁹ cell/L) and lower HSV input (or MOI), whichtypically results in a net increase of rAAV yield. After approximately52 hrs, cells and supernatant are lysed in situ and clarified by depthfiltration and absolute filtration. The clarified lysate is then beconcentrated and formulated to an appropriate buffer by Tangential FlowFiltration (TFF) prior to further purification.

Progress in the purification of AAV9 vectors.

The inventors have developed a novel AAV (e.g., AAV9) purificationprotocol based on isoelectric point (pI) precipitation. The purificationprotocol is applicable to larger scale crude lysates (>1×10⁹ cells).Using empirically established conditions, the majority of proteins incrude cell lysates generated from transfected HEK293 cells areselectively precipitated, while more than 95% rAAV9 in solution areretained. A pre-purified rAAV9 is then subjected to a 5 mL SP Sepharoseion-exchange chromatography (HiTrap SP HP, GE Healthcare), and thefractions from the elution peak are spun through Apollo 150 kD cut-offfilter. High molecular weight particles of AAV9 of are retained andconcentrated, while smaller size proteins present afterSP-chromatography are removed. This streamlined protocol results inhighly purified vector, and almost 80% of the virus originally presentin the crude lysate is retained.

Quality Control Assays.

Identity, purity, vector genome titers and infectivity titers of rAAV9preparations are assessed. Biochemical identity and purity aredetermined using polyacrylamide gel electrophoresis technique. AAVcapsid proteins VP1, 2 and 3 are visualized by silver staining orCoomassie Blue in the presence of appropriate controls. Level ofimpurities is quantified by gel imaging utilizing Quantity-One software(Bio-Rad). Vector genome titers are determined using an establishedReal-Time Quantitative PCR (Q-PCR) protocol that utilizes primers forthe SV40 polyadenylation signal sequence specific to the vector genome.Infectious Titer Assay is performed by co-infecting C12 cells (HeLacells expressing AAV2 Rep and Cap) with rAAV9 preparations and WTAdenovirus. Infected cells are subsequently trapped on nitrocellulosefilters and probed for transgene (GAA cDNA) and only cells that havebeen productively infected with rAAV produce a visible spot on film. Theratio between full and empty capsids is determined by electronmicroscopy.

EXAMPLE 11 Treatment of Neuromuscular Junction Pathology with rAAV2/9Vectors

In patients with Pompe disease, there is no contact between the nerveand acetylcholine receptors, indicating the presence of neuromuscularjunction (NMJ) pathology. As shown in FIG. 41, Gaa^(−/−) animals havemaladaptation in the neuromuscular junction and in peripheral nerves.There is an increase in the extracellular matrix and axonal loss in thesciatic nerve of Pompe mice (FIG. 42). Also, the NMJ in the diaphragmsof nine-month old wild-type and Gaa^(−/−) animals are examined. In Pompeanimals, there is a significant increase in diaphragm acetylcholinereceptor size when compared to wild-type animals (FIG. 41). Also, majoralterations are evident at the pre-synaptic membrane in the diaphragm ofGaa^(−/−) mice. Synaptotagmin is labeled to identify the pre-synapticcleft in NMJs (FIG. 43). There is a striking loss of synaptotagminexpression in Pompe animals.

Gaa^(−/−) animals are injected with AAV2/9 vectors comprising a nucleicacid molecule encoding Gaa (1×10¹¹ vg) or vehicle (see FIG. 44 forexperimental design). The treatment of Gaa^(−/−) animals with AAV2/9-GAAvectors attenuates NMJ pathogenesis in the tissue-specific models of Gaaexpression.

In a 23-month-old Gaa^(−/−) mouse, two months after the injection of theAAV2/9-DESMIN-GAA vectors directly into the right tibialis anteriormuscle, acetylcholine receptor (AChR) size in the injected leg andcontralateral leg are measured (FIG. 45). The results show a decrease inthe size of acetylcholine receptors in the leg of the Gaa^(−/−) mouseinjected with AAV2/9-DESMIN-GAA. Gaa^(−/−) animals show a significantincrease in AChR size, whereas a decrease in the size of AChR afterAAV2/9-DESMIN-GAA treatment provides positive therapeutic effectsagainst NMJ pathology. The results show that AAV2/9-GAA treatment can beused to restore or enhance neuromuscular transmission between thephrenic nerve and diaphragm. In addition to the AAV-GAA treatment,acetylcholinesterase inhibitors (ACI) can also be administered toimprove neurotransmitter release in subjects with Pompe disease.

In addition, to determine the retrograde efficiency of AAV2/9 and thepotential for reorganization of the NMJ, AAV2/9-DES-GAA vectors aredirectly injected in the tibialis anterior (TA) of Gaa^(−/−) via theintramuscular route. FIG. 46 shows the loss of NMJ pre- andpost-synaptic integrity in affected (Gaa^(−/−)) animals compared towild-type. FIG. 46 also shows the reorganization of the NMJ in a Pompeanimal following intramuscular AAV2/9-DES-GAA administration.Longitudinal sectioning of the corresponding AAV2/9-GAA sciatic nervereveals an increased signal in growth associated protein 43 (Gap43)labeling (FIG. 47). Gap43 has been associated with axonal regeneration,long-term potentiation, and is a crucial component of the pre-synapticterminal. The positive treatment effects are mediated by high vectorgenome copy number and expression of GAA at the site of injection (TA)and in the lumbar spinal cord.

As shown in FIG. 48, intraspinal injections (C3-C5) of AA2/5V-GAA areperformed to Gaa^(−/−) animals to target the phrenic motor neuron pool.Vector treated animals demonstrate high levels of GAA at the site ofinjection, reduction of glycogen, and improved respiratory function tolevels near wild-type values (FIG. 48).

OTHER EMBODIMENTS

Any improvement may be made in part or all of the compositions andmethod steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. This invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contraindicated by context.

1. A method of improving impaired neuromuscular junction integrity,comprising administering, to a subject with impaired neuromuscularjunction integrity, an effective amount of a composition comprising arAAV 2/9 vector, wherein the rAAV2/9 vector comprises a heterologousnucleic acid molecule operably linked to a promoter, and the therapeuticcomposition is administered to the subject via intramuscular,intrathoracic, intraspinal, intracisternal, intrathecal, or intravenousinjection.
 2. The method according to claim 1, wherein the heterologousnucleic acid molecule encodes acid α-glucosidase (GAA).
 3. The methodaccording to claim 1, wherein the promoter is a cytomegalovirus (CMV)promoter, a desmin (DES) promoter, a synapsin I (SYN) promoter, or amuscle creatine kinase (MCK) promoter.
 4. The method according to claim1, wherein the subject has impaired neuromuscular caused by aneuromuscular disease.
 5. The method according to claim 1, wherein thesubject has impaired neuromuscular caused by a disease selected from thegroup consisting of Pompe disease, amyotrophic lateral sclerosis, spinalmuscular atrophy, multiple sclerosis, glycogen storage disease type 1a,limb Girdle muscular dystrophy, Barth syndrome, and myasthenia gravis.6. The method according to claim 1, wherein the subject is a human. 7.The method, according to claim 2, further comprising administering anacetylcholinesterase inhibitor (ACI) to the subject.
 8. A method oftreating a neuromuscular disease, comprising administering, to a subjectin need of such treatment, an effective amount of a compositioncomprising a rAAV 2/9 vector, wherein the rAAV2/9 vector comprises aheterologous nucleic acid molecule operably linked to a promoter, andthe therapeutic composition is administered to the subject viaintramuscular, intrathoracic, intraspinal, intrathecal, intracisternal,or intravenous injection.
 9. The method according to claim 8, whereinthe neuromuscular disease is selected from the group consisting of Pompedisease, amyotrophic lateral sclerosis, spinal muscular atrophy,multiple sclerosis, glycogen storage disease type 1a, limb Girdlemuscular dystrophy, Barth syndrome, and myasthenia gravis
 10. The methodaccording to claim 9, wherein the neuromuscular disease is Pompedisease.
 11. The method according to claim 8, wherein the heterologousnucleic acid molecule encodes acid α-glucosidase (GAA).
 12. The methodaccording to claim 8, wherein the promoter is a cytomegalovirus (CMV)promoter, a desmin (DES) promoter, a synapsin I (SYN) promoter, or amuscle creatine kinase (MCK) promoter.
 13. The method according to claim8, wherein the subject is a human.
 14. The method, according to claim11, further comprising administering an acetylcholinesterase inhibitor(ACI) to the subject.
 15. A method of improving impaired neuromuscularjunction integrity and/or for treatment a neuromuscular disease,comprising administering to a subject an effective amount of acomposition comprising a rAAV vector, wherein the rAAV vector comprisesa heterologous nucleic acid molecule operably linked to a promoter,wherein the subject has impaired neuromuscular junction integrity and/ora neuromuscular disease and the subject does not have Pompe disease. 16.The method according to claim 15, wherein the rAAV vector is selectedfrom a rAAV2/1, rAAV2/8, or rAAV2/9 vector.
 17. The method according toclaim 15, wherein the therapeutic composition is administered to thesubject via intramuscular, intrathoracic, intraspinal, intracisternal,or intravenous injection.
 18. The method according to claim 15, whereinthe promoter is a cytomegalovirus (CMV) promoter, a desmin (DES)promoter, a synapsin I (SYN) promoter, or a muscle creatine kinase (MCK)promoter.
 19. The method according to claim 15, wherein the subject hasa neuromuscular disease selected from the group consisting ofamyotrophic lateral sclerosis, spinal muscular atrophy, multiplesclerosis, glycogen storage disease type 1a, limb Girdle musculardystrophy, Barth syndrome, and myasthenia gravis.
 20. The methodaccording to claim 15, wherein the subject is a human.